Embedded chimeric peptide nucleic acids and uses thereof

ABSTRACT

The present invention relates to an ecPNA having the general structure: 
       H 2 N—X—B—Y—COOH
 
     and uses thereof, wherein X is A or C and Y is A or C with the proviso that when X is A, Y is C, and when X is C, Y is A; A represents an oligopeptide structure, the sequence of which comprises a sequence which renders the compound able to enter the nucleus of a cell; B represents a peptide nucleic acid (PNA) structure at least 12 nucleotides in length, the sequence of which is capable of hybridizing with a DNA within the nucleus of the cell, which DNA is within a promoter region of a gene; C represents an oligopeptide structure; and each — represents a chemical linkage between the structures at each side thereof, which may be the same as or different from each other such linkage. The ecPNAs provided herein can upregulate or repress gene transcription and are useful for treating diseases requiring changes in transcription and for induction of inducible pluripotent stem (iPS) cells.

This application is a continuation of U.S. Ser. No. 13/585,554, filedAug. 14, 2012, now U.S. Pat. No. 8,927,502, issued Jan. 6, 2015, whichis a continuation-in-part of PCT International Application No.PCT/US2011/025128, filed Feb. 16, 2011, which claims priority of U.S.Provisional Application No. 61/338,316, filed Feb. 16, 2010; and acontinuation-in-part of PCT International Application No.PCT/US2011/062423, filed Nov. 29, 2011, which claims priority of U.S.Provisional Application Nos. 61/534,240, filed Sep. 13, 2011, and61/417,760, filed Nov. 29, 2010, the contents of each of which arehereby incorporated by reference in their entireties.

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“141215_(—)0028_(—)82577-CA_Substitute_Sequence_Listing_AHC.TXT”, whichis 39.1 kilobytes in size, and which was created Dec. 15, 2014 in theIBM-PC machine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Dec. 15, 2014 aspart of this application.

Throughout this application, certain publications are referenced inbrackets. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order todescribe more fully the state of the art to which this inventionrelates.

The research leading to the present invention was supported, in part, bygrant number HL073437, awarded by the National Institutes of Health.Accordingly, the U.S. Government has certain rights in this invention.

TECHNICAL FIELD

The present invention provides novel embedded chimeric peptide nucleicacid molecules (ecPNAs) comprising (i) a PNA targeting a promoter regionof a gene, wherein said PNA is conjugated to (ii) at least one celland/or nuclear entry sequence and (iii) a transcription activation orrepression domain. The ecPNAs of the invention can modulate geneexpression and are useful for treating diseases requiring changes intranscription and for induction of inducible pluripotent stem (iPS)cells.

BACKGROUND OF INVENTION

Detection of DNA sequences such as DNA sequences within genes has beenused for various purposes including research and disease diagnosis.Compounds useful for detecting DNA sequences include DNA and RNAoligonucleotide probes, typically labeled with some detectable markersuch as a radioisotope, a fluorescent dye, or an immunogenic peptide.Nevertheless, there is a continuing need for compounds which can beemployed for such purposes. The compounds of the present inventionfulfill this need.

Certain of the compounds of this invention may also be used to regulatetranscription and expression of genes including genes the regulatedexpression of which is implicated in, or can be useful in, the treatmentor amelioration of disease.

Correct regulation of gene expression is required for fundamentalprocesses in differentiation and development. [See Davidson, E. H., GeneActivity in Early Development, Edn. Third. (Academic Press, Inc.,Orlando; 1986)]. In a number of cases, the transcriptional onset anddecline of a series of closely related genes are tightly andsequentially controlled, a process that is critical for attaining thecorrect genotypic readout and proper phenotypic effect. [See Bresnick,E. H., Martowic, M. L., Pal, S. & Johnson, K. D., Developmental controlvia GATA factor interplay at chromatin domains. J Cell Physiol 205, 1-9(2005), Stamatoyannopoulos, G., The molecular basis of blood diseases,Edn. 3rd. (W.B. Saunders, Philadelphia; 2001); and Krumlauf, R,Noordermeer D., de Laat W., Joining the loops: β-globin gene regulation.IUMMB Life 60, 824-833 (2008); and Krumlauf, R., Hox genes in vertebratedevelopment. Cell 78, 191-201 (1994).]A particularly well-studiedexample of this process is the developmental control of the hemoglobinproteins, particularly those encoded by the genes within the β-likeglobin locus. [See Stamatoyannopoulos, G., The molecular basis of blooddiseases, Edn. 3rd. (W.B. Saunders, Philadelphia; 2001); Krumlauf, R.,Hox genes in vertebrate development. Cell 78, 191-201 (1994); andSchechter, A. N., Hemoglobin research and the origins of molecularmedicine. Blood 112, 3927-3938 (2008).] These variants exhibit asequential erythroid-restricted pattern of expression duringdevelopment, beginning with the yolk sac ε-globin, switching to thefetal γ-globin, and ending with the adult β-globin.

The critical requirement for correct regulation of this locus isdemonstrated by the moderate to life-threatening clinical manifestationsexhibited by the β-thalassemias. β-thalassemia is primarily caused bymutations in the β-globin gene that lead to reduced or complete loss ofβ-globin expression. Along with other hemoglobinopathies (such as sicklecell disease), they give rise to the most common single gene geneticdisorder worldwide. [See Weatherall, D. J., in The Molecular Bases ofBlood Diseases. (eds. G. Stamatoyannopoulos, A. W. Nienhuis, P. W.Majerus & H. Varmus) 207-205 (W.B. Saunders Co., Philadelphia; 1994).]Pharmacological reactivation of the silent fetal (γ□) globin chainprovides a therapeutic benefit to these patients by compensating forabsent adult β-globin chains (in β-thalassemia) or by interfering withthe polymerization of mutant hemoglobins (in sickle cell disease);however, these are not always free from complications. [See WeatherallD. J., in The Molecular Bases of Blood Diseases. (eds. G.Stamatoyannopoulos, A. W. Nienhuis, P. W. Majerus & H. Varmus) 157-256(W. B. Saunders Co., Philadelphia; 1994); and Atweh, G. F. & Schechter,A. N., Pharmacologic induction of fetal hemoglobin: raising thetherapeutic bar in sickle cell disease. Curr Opin Hematol 8, 123-130(2001).] As a result, there remain compelling reasons to search fornovel approaches and reagents that achieve reactivation with lowtoxicity and high penetrance.

Regulation of gene expression is also of particular importance for thegeneration of induced pluripotent stem (iPS) cells. iPS cells arepluripotent stem cells expressing many of the genetic and phenotypiccharacteristics of embryonic stem (ES) cells. iPS cells have the samegross morphology as ES cells, proliferative properties, form teratomasafter transplantation into nude mice, and have the ability todifferentiate along all 3 germ layers in vitro. Their responses to keyfactors such as retinoic acid and leukemia inhibitory factor (LIF) arealso the same as those observed for ES cells [Okita et al, Generation ofgermline-competent induced pluripotent stem cells. Nature 448: 313-317(2007)]. Generation of iPS cells is useful for both in vitro study ofstem cells (e.g., factors controlling stem cell differentiation) and forthe application of iPS cells for the treatment of disease. For example,the ability to reprogram cells from human blood would be useful for thegeneration of patient-specific stem cells for treatment of diseases inwhich the disease-causing somatic mutations are restricted to cells ofthe hematopoietic lineage.

Currently, methods for generating iPS cells from somatic cells arelimited, and novel methods are needed. iPS cells have the potential torevolutionize medicine, as they can theoretically generate anydifferentiated tissue from the self-same individual and thus avoidtissue rejection and other complications. An original protocol forgenerating iPS cells established iPS cells from murine and humanfibroblasts by introducing four specific transcription factors, SOX2,OCT4, KLF4, and c-MYC, into the cells by viral transduction. [See,Lowry, W. E., et al. Generation of human induced pluripotent stem cellsfrom dermal fibroblasts. Proc. Natl. Acad. Sci. USA 105, 2883-2888(2008); Maherali, N., et al. Directly reprogrammed fibroblasts showglobal epigenetic remodeling and widespread tissue contribution. CellStem Cell; 1, 55-70 (2007); Park, et al. Reprogramming of human somaticcells to pluripotency with defined factors. Nature 451, 141-146 (2008);Takahashi, K., and Yamanaka, S. Induction of Pluripotent Stem cells fromMouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell,126, 663-676 (2006); Takahashi, K. et al. Induction of Pluripotent StemCells from Adult Human Fibroblasts by Defined Factors. Cell 131, 861-872(2007); Yu, J. et al. Induced pluripotent stem cell lines derived fromhuman somatic cells Science; 318, 1917-1920 (2007); Stadtfeld andHochedlinger, Induced pluripotency: history, mechanisms, andapplications. Genes Dev; 24:2239-2263 (2010); and Hochedlinger andPlath, Epigenetic reprogramming and induced pluripotency. 136; 509-523(2009).]

Recently, iPS cells were generated from CD34+ mobilized human peripheralblood cells using retroviral transduction of OCT4, SOX2, KLF4, and c-MYC[see, Loh, Y. et al. Generation of induced pluripotent stem cells fromhuman blood. Blood; 113(22):5476-5479 (2009)], and from CD133+ humancord blood (CB) cells [see, Giorgetti, A. et al. Generation of inducedpluripotent stem cells from human cord blood using OCT4 and SOX2. CellStem Cell; 5:353-357 (2009)]. iPS cells were generated from CD133+CBcells by retroviral transduction of CB cells with OCT4 and SOX2. CBcells are considered an alternative to bone marrow (BM) as a source ofhematopoietic stem cells for transplantation. CB cells can be collectedwithout any risk for the donor, are young cells expected to carryminimal somatic mutations, and possess the immunological immaturity ofnewborn cells [see, Rocha et al., et al. Transplants of Umbilical-CordBlood or Bone Marrow from Unrelated Donors in Adults with AcuteLeukemia. N. Engl. J. Med. 351, 2276-2285 (2004)]. These propertiesallow for less stringent criteria for HLA-donor-recipient selection,which represents a decisive benefit for transplantation and has resultedin more than 400,000 immunologically characterized CB units beingcurrently available worldwide through a network of CB banks [see,Gluckman, E., and Rocha, V. Cord blood transplantation: state of theart. Haematologica; 94, 451-454 (2009)].

These approaches brought closer the possibility of usingpatient-specific cells in cell-based therapy. At least three limitationsof those methods, however, were immediately recognizable: (i) the geneswere transduced virally; (ii) at least one of the factors, c-Myc, is aknown oncogene; and (iii) the process was inefficient. In particular, inthe above described studies, retroviral transduction was used tointroduce the genes required for iPS cell generation. The use of virusesto deliver the reprogramming factors entails permanent geneticalterations that render the cells inappropriate for many in vitro and invivo applications. Retroviral gene transduction is associated with anumber of limitations (low efficiency), and potential danger for use inhuman therapy, including the risk of production of replication competentvirus, which can infect humans [reviewed in Kurian, K. M. et al. (2000)J Clin Pathol: Mol Pathol; 53:173-176; Stadtfeld and Hochedlinger (2010)Genes Dev; 24:2239-2263].

Thus, what is needed in the art are novel, efficient ways for inducing aselected target gene or genes in a cell without the use of retroviraltransduction and without genetically perturbing the recipient cell.

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which thephosphodiester backbone of DNA is replaced by an achiral unchargedpolyamide backbone. [See Nielsen, P. E., Egholm, M., Berg, R. H. &Buchardt, O., Sequence-selective recognition of DNA by stranddisplacement with a thymine-substituted polyamide. Science 254,1497-1500 (1991).] They are true DNA mimics, as they form Watson-Crickbonds with DNA and RNA, but are of higher thermal stability than naturalduplexes due to the lack of electrostatic repulsion. [See Kaihatsu, K.,Janowski, B. A. & Corey, D. R., Recognition of chromosomal DNA by PNAs.Chem Biol 11, 749-758 (2004).] They are also resistant to proteases andnucleases, and thus afford a significantly greater biological stabilityin culture and in vivo. [See Pooga, M., Land, T., Bartfai, T. & Langel,U., PNA oligomers as tools for specific modulation of gene expression.Biomol Eng 17, 183-192 (2001).]A unique aspect of PNAs is that aminoacids can be covalently added to the peptide backbone at either end ofthe sequence of bases. The PNA/DNA interaction may occur through triplehelical (Hoogsteen) base paring (PNA/DNA/PNA) or via a single strandinvasion. [See Kaihatsu, K., Janowski, B. A. & Corey, D. R., Recognitionof chromosomal DNA by PNAs. Chem Biol 11, 749-758 (2004); and Zhang, X.,Ishihara, T. & Corey, D. R., Strand invasion by mixed base PNAs and aPNA-peptide chimera. Nucleic Acids Res 28, 3332-3338 (2000).] In asingle strand invasion, PNA, which can be of mixed sequence design,hybridizes with one strand of DNA through Watson-Crick base pairing andsimply replaces the other strand of the double helix.

PNA molecules are thus promising candidates for clinical use as agentsto modulate gene expression. For the most part, PNAs have been used asan antigene agent because they have the capacity for down-regulatinggene expression in cultured cells and in animals. [See Nielsen, P. E.,Peptide nucleic acids as antibacterial agents via the antisenseprinciple. Expert Opin Investig Drugs 10, 331-341 (2001); Cutrona, G. etal., Effects in live cells of a c-myc anti-gene PNA linked to a nuclearlocalization signal. Nat Biotechnol 18, 300-303 (2000); Janowski, B. A.et al. Inhibiting transcription of chromosomal DNA with antigene peptidenucleic acids. Nat Chem Biol 1, 210-215 (2005); Hu, J. et al.,Allele-specific silencing of mutant huntingtin and ataxin-3 genes bytargeting expanded CAG repeats in mRNAs. Nat Biotechnol 27, 478-484(2009); and Nielsen, P. E., PNA Technology. Mol Biotechnol 26, 233-248(2004).]

PNA molecules have also been used as probes for targeted nucleic acidbinding, and to follow the subcellular trafficking of plasmid DNA.Additionally, conjugation of markers such as fluorophores to PNAmolecules has been described for these purposes. [See also Zhilina etalt, Peptide Nucleic Acid Conjugates: Synthesis, Properties andApplications. Current Topics in Medicinal Chemistry 4:1119-1131 (2005)].PNA molecules have also been utilized for single base pair mutationanalysis by PNA directed PCR clamping. [See also Orum et al., Singlebase pair mutation analysis by PNA directed PCR clamping. Nucleic AcidsResearch 21(23):5332-5336 (1993)] However, there is a need for improvedPNA structures capable of efficiently detecting the presence of DNAwithin the nucleus of cells.

PNAs have been modified to maximize cellular/nuclear entry in order toincrease the efficiency for in vivo applications. [See Cutrona, G. etal., Effects in live cells of a c-myc anti-gene PNA linked to a nuclearlocalization signal. Nat Biotechnol 18, 300-303 (2000); Nielsen, P. E.,Addressing the challenges of cellular delivery and bioavailability ofpeptide nucleic acids (PNA). Q Rev Biophys 38, 345-350 (2005); andBraun, K. et al, A biological transporter for the delivery of peptidenucleic acids (PNAs) to the nuclear compartment of living cells. J MolBiol 318, 237-243 (2002).] However, currently there is no establishedPNA conjugated system that combines these varied modifications and hasbeen demonstrated to stably and efficiently enter, target, andtranscriptionally activate an endogenous chromosomal locus in livingcells.

Thus, there remains a great need in the art for novel, efficient andnon-toxic compounds which can affect gene expression. Such compoundswould be particularly useful in treatment of β-globin disorders, whichcan be treated by upregulating γ•-globin transcription in bone marrowcells, and for the generation of iPS cells, for example by the inductionof genes such as OCT4, SOX2, c-MYC, and/or KLF4.

SUMMARY OF THE INVENTION

The present invention addresses these and other needs by providing novelembedded chimeric peptide nucleic acid (ecPNA) molecules.

The present invention relates to an ecPNA having the general structure:

H₂N—X—B—Y—COOH

and uses thereof, wherein X is A or C and Y is A or C with the provisothat when X is A, Y is C, and when X is C, Y is A; wherein A representsan oligopeptide structure, the sequence of which comprises a sequencewhich renders the ecPNA able to enter the nucleus of a cell; wherein Brepresents a peptide nucleic acid (PNA) structure at least 12nucleotides in length, the sequence of which is capable of hybridizingwith a DNA within the nucleus of the cell, which DNA is within apromoter region of a gene; wherein C represents an oligopeptidestructure; and wherein each — represents a chemical linkage between thestructures at each side thereof, which may be the same as or differentfrom each other such linkage.

The present invention also relates to an ecPNA having the generalstructure:

H₂N—X—B—Y—COOH

and uses thereof, wherein X is D or C and Y is D or C with the provisothat when X is D, Y is C, and when X is C, Y is D; wherein D representsa compound which renders the ecPNA able to bind to a receptor on a cell;wherein B represents a peptide nucleic acid (PNA) structure at least 12nucleotides in length, the sequence of which is capable of hybridizingwith a DNA within the nucleus of the cell, which DNA is within apromoter region of a gene; wherein C represents an oligopeptidestructure; and wherein each — represents a chemical linkage between thestructures at each side thereof, which may be the same as or differentfrom each other such linkage.

Aspects of the present invention provide a method for generating aninducible pluripotent stem (iPS) cell from a source cell comprisingadministering to the source cell an effective amount for inducing theiPS cell of at least one of the ecPNA molecules of the invention,wherein the ecPNA molecule upregulates transcription of a target geneselected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28,NANOG, PRDM14, and NFRKB. In certain embodiments, the source cell isselected from the group consisting of a CD34+ peripheral blood cell, aCD34+ bone marrow cell, a CD133+ cord blood cell, a neural cell and afibroblast.

In some embodiments, the ecPNA molecule for generating iPS cellcomprises a PNA molecule having a nucleic acid sequence that iscomplementary to a sequence comprised in the 200 base pair (bp) regionupstream of the transcription start site of the target gene. In someembodiments, the PNA molecule is about 12 to about 15 nucleotides (nt)in length.

In yet other embodiments, the method for generating an iPS cellcomprises administering at least two ecPNA molecules according to thepresent invention.

In certain of the above embodiments for methods for generating an iPScell, one of the two ecPNA molecules upregulates transcription of OCT4gene. In yet other embodiments, one of the two ecPNA moleculesupregulates transcription of SOX2 gene. In still other embodiments, oneof the two ecPNA molecules upregulates transcription of OCT4 gene andthe other of the two ecPNA molecules upregulates transcription of SOX2gene.

In certain of the above embodiments, the source cell is a human cell.

In some of the above embodiments, the method for generating an iPS cellfurther comprises conjoint administration of a second agent selectedfrom the group consisting of hydroxyurea, a short chain fatty acid(SCFA) inducer, 5-azacytidine, and a histone deacetylase inhibitor. Incertain embodiments, the short chain fatty acid (SCFA) inducer isbutyrate. In other embodiments, the histone deacetylase inhibitor issuberoylanilide hydroxamic acid (SAHA).

The invention is described in detail below, with reference to theaccompanying figures and by way of non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict in vitro target binding specificity of four PNAsdesigned to interact with the human γ-globin promoter. In FIG. 1A,PNA150 (SEQ ID NO: 4), PNA116 (SEQ ID NO: 3), PNA78 (SEQ ID NO: 1), andPNA7 (SEQ ID NO: 2) were designed to target the proximal promoter region(−202 to +33) of the human fetal γ-globin gene at specific sitesindicated by the filled in boxes. The relative location of knownpromoter elements within this region is also indicated. Orientation ofthe PNA molecule relative to the promoter sequence is shown above, witha lys at its 3′-end and a biotin molecule (circle) attached at the5′-end. In FIG. 1B, different concentrations of PNAs (0-10 μM) wereincubated with a constant amount of radioactively labeled DNAoligonucleotide (10 nM) containing either the wild type (wt) or mutated(mut) target sequence followed by incubation with streptavidin Dynabeadsand collection with a magnetic particle concentrator. Bound/input ratioswere obtained after scintillation counter analysis. In FIG. 1C,permanganate probing confirms the specificity of PNA78 by showing thatoxidized and cleaved T bases (lane 3) are accessible within thedesignated target sequence compared to ‘no PNA’ (--) and PNA7 (asnon-interacting PNA) controls. The A+G sequence ladder is shown in lane1, along with the nucleotide sequence of the γ-globin promoter alignedon the left.

FIGS. 2A-C depict cellular and nuclear localization of PNA78 and itsderivatives. All PNA molecules contained a biotin tag at their 5′-endand were visualized with streptavidin-FITC. FIG. 2A depicts fluorescentmicroscopy showing uptake of PNA78 by K562 cells exposed to differentconcentrations as indicated. PNA signals appear as bright punctate dots.FIG. 2B depicts fluorescent microscopy showing uptake of PNA78/TAT orPNA78/NLS (at final concentrations of 10 uM) by COS7 cells. Live cell(unfixed) images: left, DIC (brightfield) image; right, fluorescence(FITC) indicating positive PNA signals. FIG. 2C depicts confocalmicroscopy showing uptake of PNA78/TAT, PNA78/NLS, or ‘TAT alone’(positive control) (all at a final concentration of 10 uM) into K562cells that had been attached to poly-L-lysine coated slides. Live cell(unfixed) images: left, DIC image; right, green fluorescence (FITC)indicating positive PNA signals.

FIGS. 3A-B depict in vivo target binding of PNA78/TAT in K562 cells.Specifically, FIG. 3A depicts steps of modified chromatin associationassay using streptavidin-conjugated magnetic beads and the magneticparticle concentrator. During the actual experiment, a portion of thetreated cells were simultaneously analyzed for PNA uptake by confocalmicroscopy. FIG. 3B, left panel, depicts a confocal image showingnuclear-entering efficiency of WT PNA78/TAT and MUT PNA78/TAT. ‘No PNA’and ‘TAT alone’ were included as negative and positive controls. DRAQ5was used as a nuclear stain for live (unfixed) cells. FIG. 3B, rightpanel, depicts quantitative chromatin association analysis of K562 cellsafter exposure to WT PNA78′TAT or MUT PNA78/TAT. ‘No PNA’ and ‘TATalone’ served as controls. The results of quantitative analysis of theγ-globin promoter DNA in the precipitated material is shown.

FIGS. 4A-C depict in vivo transcriptional activity of chimeric PNA78derivatives. In FIG. 4A, a transient assay was performed in K562 cellsthat had been transfected with plasmid containing the γ-globin promoter(−299˜+36) upstream of the luciferase reporter (shown below) followed byexposure to PNA78/TAT, PNA78/NLS, TAT alone, or buffer. Activity valueswere normalized to a cotransfected Renilla control. In FIG. 4B, K562cells were transiently transfected with a luciferase reporter containingeither wild type (wt) γ-globin promoter sequence or one that was mutatedat the PNA target region (mut) (AATAGTTTTATAGTA; SEQ ID NO: 5), followedby exposure to PNA78/TAT or buffer. Values were normalized to acotransfected Renilla control. In FIG. 4C a reporter plasmid bearing 4repeats of the PNA78 target sequence and the minimal SV40 promoterupstream of luciferase gene (schematic shown below; PNA78 sequence isunderlined) was transfected into either K562 or 293T cells and followedby exposure to PNA78/TAT, VP2/PNA78/TAT, or buffer. Values werenormalized to a cotransfected Renilla control.

FIGS. 5A-C show that PNA78 conjugated to a minimal activation domainreactivates dormant γ globin expression in vivo in mouse adult bonemarrow cells (MBC) containing the human βYAC. Cells were treated witheach of four modified PNA78 molecules (PNA78/TAT, VP2/PNA78/TAT,Bio-ATF/PNA78/TAT, ATF-Bio/PNA78/TAT), ‘TAT alone’, or buffer (‘no PNA’)and analyzed by confocal microscopy (a) or by quantitative RT-PCR (b) 16hours after PNA treatment. FIG. 5A depicts a confocal image of MBC-βYACshowing nuclear-entering efficiency. FIG. 5B shows an expression profileof γ globin levels in MBC-βYAC cells in (a) analyzed by quantitativeRT-PCR with 2 sets of primers as described in Methods. Each experimentis the average of triplicate sample analyses. In FIG. 5C MBC-βYAC cellswere treated with buffer (‘no PNA’) or ATF-Bio/PNA78/TAT and analyzed byconfocal microscopy 36 hours later by fixing and staining with DAPI(blue) and an anti-human γ-globin protein antibody linked to Alexa 568(red). K562 cells were also stained and served as a positive control forγ-globin protein detection (arrowheads).

FIG. 6 shows persistence of ATF-Bio/PNA78/TAT) after introduction intocells. In the figure, a series of confocal images are shown of K562cells that have been exposed to ATF-Bio/PNA78/TAT at day 0.Visualization of PNA was with streptavidin-FITC, and DRAQ5 was used as anuclear stain for live (unfixed) cells. (--) are cells that had not beenexposed to PNA. In some cases the same field was monitored on successivedays. Each panel contains: top left, fluorescence (FITC) positive PNAsignal; top right, DRAQ5 nuclear signal; bottom left, merged DRAQ5/FITCsignal.

FIGS. 7-10 show the nucleic acid and amino acid sequences of human OCT4(FIG. 7) (SEQ ID NO: 64 and SEQ ID NO: 65), SOX2 (FIG. 8) (SEQ ID NO: 66and SEQ ID NO: 67), KLF4 (FIG. 9) (SEQ ID NO: 68 and SEQ ID NO: 69), andc-MYC (FIG. 10) (SEQ ID NO: 70 and SEQ ID NO: 71). The bolded,underlined nucleic acid residues are the 200 bp region upstream of thetranscription start site. This 200 bp region is the region targeted bythe ecPNA molecules of the invention. The start codon, ATG isunderlined, bolded and italicized and the transcription start site isitalicized.

FIGS. 11A-B show the analysis of human peripheral blood CD34+ cells atdifferent time points after induction of erythroid differentiation byerythropoietin (Epo). FIG. 11A is a line graph showing the percent ofthe cell culture expressing the cell surface markers CD34, CD235a, CD36,and CD71 at the indicated time points (days, “D”). FIG. 11B containsimages showing the morphological (Giemsa) analyses performed on humanperipheral blood CD34+ samples removed at the indicated day of culture.The progressive changes toward a more terminally differentiated statesuch as later stage erythroblasts are indicated by arrows and matureenucleated red cells are indicated by asterisks.

FIGS. 12A-C show the analysis of human peripheral blood CD34+ cellsduring the SCF/Epo phase of culture. FIG. 12A contains dot plots showingflow cytometric profiles of CD36 and CD235a expression in humanperipheral blood (H-BP) samples at Day 2 and Day 4 of incubation inSCF/Epo, showing increases in their expression during culture. FIG. 12Bis a fluorescent image showing beta globin expression in H-PB CD34+cells and FIG. 12C is an expression analysis by semi-quantitative RT-PCRof β-globin mRNA in H-PB cells on Day 0, 2, 4, 5, and 7 as indicated.

FIGS. 13A-C are an analysis of the effect of PNA78 conjugated to aminimal activation domain on activating γ globin expression indifferentiating adult human peripheral blood (H-PB) CD34+ cells. FIG.13A is a bar graph showing the mRNA level of γ globin in cultured CD34+cells 16 hours after treatment with PNA78/TAT, ATF-Bio/MUT-PNA78/TAT, orATF-Bio/WT-PNA78/TAT. Analysis of triplicates was performed byquantitative RT-PCR (*p<0.005). FIG. 13B contains images showingimmunostaining and analysis by confocal microscopy of γ globin proteinin cultured CD34+ cells from the same experiment as in FIG. 13A,performed after treatment with buffer (‘no PNA’) or chimeric PNA78molecules. K562 cells served as a positive control. FIG. 13C containsdot plots and histograms from toxicity analysis by flow cytometry ofcultured CD34+ cells after PNA treatment. The effectiveness of PNA78entry was monitored with streptavidin-FITC.

FIGS. 14A-C are an expression profile of erythroid markers in humanperipheral blood CD34+ cells after PNA treatment. Human peripheral blood(H-PB) CD34+ cells from the same experiment as in FIG. 8 (day 2 ofculture) were analyzed by flow cytometry 24 hours after treatment withbuffer (‘no PNA’), PNA78/TAT, ATF-Bio/MUT-PNA78/TAT, orATF-Bio/WT-PNA78/TAT. FIG. 14A shows forward scatter (“FSC”) and sidescatter (“SSC”) profiles of the indicated cells. FIG. 14B shows the CD34and CD36 expression profiles of the cells and FIG. 14C shows the CD36and CD235a expression profiles of the indicated cells.

FIGS. 15-18 show the nucleic acid and amino acid sequences of humanLIN28 (FIG. 15) (SEQ ID NO: 72 and SEQ ID NO 73), NANOG (FIG. 16) (SEQID NO: 74 and SEQ ID NO: 75), PRDM14 (FIG. 17) (SEQ ID NO: 76 and SEQ IDNO: 77) and NFRKB (FIG. 18) (SEQ ID NO: 78 and SEQ ID NO: 79). Thebolded, underlined nucleic acid residues are the 200 bp region upstreamof the transcription start site. This 200 bp region is the regiontargeted by the ecPNA molecules of the invention. The start codon ATG isunderlined, bolded and italicized and the transcription start site isitalicized.

FIG. 19 shows the in vitro target binding specificity of four PNAsdesigned to interact with the human SOX2 promoter. Sox2-2, -43, -62, and-81 target the proximal promoter region (-1 to -200) of the human SOX2gene at specific sites. All PNAs contain a biotin molecule attached attheir 5′-end. PNAs were incubated with radioactively labeled DNAoligonucleotide containing either the wild type or mutated targetsequence followed by incubation with streptavidin Dynabeads andcollection with a magnetic particle concentrator. Bound/inputpercentages were determined after scintillation counter analysis. Arrowsindicate molecules selected for further study.

FIG. 20 shows the in vitro target binding specificity of four PNAsdesigned to interact with the human OCT4 promoter. Oct4-54, -83, -100,and -111 target the proximal promoter region (-1 to -200) of the humanOCT4 gene at specific sites. Analyses were performed as in FIG. 19.

FIG. 21 shows a schematic of ecPNA molecules. Top is the general ecPNAlayout as described herein. Immediately below is the TAT-PNA78-ATFactivation molecule as used in the PNAS manuscript. Below this are thefour activation variants containing either SOX2- or OCT4-specific PNAsequences as described in the text (part A). Below this is therepression molecule containing the FOG12 repression motif as describedin the text (part B). Finally, below this is the Epo mimetic (epoM)design described in the text (part C). All molecules contain biotin forease of detection (not show).

FIG. 22 shows the delivery of TAT-PNA78-FOG12 to K562 cells. Confocalfluorescence microscopy of live K562 cells attached to poly-1-lysinecoated is shown. (A) Brightfield picture of cells showing DRAQ5-stainednuclei (red). (B) Presence of intracellular TAT-PNA78-FOG12, visualizedby strepavidin-FITC staining (green). (C) Overlay of panels A and B(nuclear and TAT-PNA78-FOG12 staining) shows yellow in areas of overlap(that is, successful entry of TAT-PNA78-FOG12 in the nucleus). Controlexperiments (cells not exposed to TAT-PNA78-FOG12) do not show anyyellow overlap color (not shown).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an ecPNA having the general structure:

H₂N—X—B—Y—COOH

and uses thereof, wherein X is A or C and Y is A or C with the provisothat when X is A, Y is C, and when X is C, Y is A; wherein A representsan oligopeptide structure, the sequence of which comprises a sequencewhich renders the ecPNA able to enter the nucleus of a cell; wherein Brepresents a peptide nucleic acid (PNA) structure at least 12nucleotides in length, the sequence of which is capable of hybridizingwith a DNA within the nucleus of the cell, which DNA is within apromoter region of a gene; wherein C represents an oligopeptidestructure; and wherein each — represents a chemical linkage between thestructures at each side thereof, which may be the same as or differentfrom each other such linkage.

In some embodiments, X is A and Y is C. In other embodiments, X is C andY is A.

The present invention also relates to an ecPNA having the generalstructure:

H₂N—X—B—Y—COOH

and uses thereof, wherein X is D or C and Y is D or C with the provisothat when X is D, Y is C, and when X is C, Y is D; wherein D representsa compound which renders the ecPNA able to bind to a receptor on a cell;wherein B represents a peptide nucleic acid (PNA) structure at least 12nucleotides in length, the sequence of which is capable of hybridizingwith a DNA within the nucleus of the cell, which DNA is within apromoter region of a gene; wherein C represents an oligopeptidestructure; and wherein each — represents a chemical linkage between thestructures at each side thereof, which may be the same as or differentfrom each other such linkage.

In some embodiments, X is D and Y is C. In other embodiments, X is C andY is D.

In some embodiments, D comprises a polypeptide sequence that binds to areceptor on a cell.

In some embodiments, D binds to the erythropoietin (Epo) receptor. Insome embodiments, D is an analogue of Epo. In some embodiments D is anEpo mimetic peptide.

In some embodiments, the gene is a gene, the regulation of thetranscription and expression of which is desired.

In certain embodiments, the oligopeptide structure C is detectable whenthe ecPNA is bound to the DNA.

In some embodiments, the sequence of C comprises a sequence whichrenders the ecPNA able to regulate the transcription and expression ofthe gene.

In certain embodiments, each — may be a chemical bond or a chemicallinker.

In some embodiments, at least one — is a chemical bond.

In certain embodiments, the chemical bond is a covalent bond, an amidebond, or a peptide bond.

In some embodiments, at least one — is a chemical linker.

In some embodiments of the invention, the chemical linker comprises anamino acid, biotin, an ether (O), a stable polyether (OO), AEEA(2-aminoethoxy-2-ethoxyacetic acid), or a cleavable disulfide linkage.

In some embodiments of the invention, the sequence of C renders theecPNA able to regulate the transcription and expression of the gene byactivating transcription of the gene.

In some embodiments of the invention, the sequence of C renders theecPNA able to regulate the transcription and expression of the gene byupregulating transcription of the gene.

In some embodiments, the sequence of C renders the ecPNA able toregulate the transcription and expression of the gene by downregulatingthe transcription of the gene.

In some embodiments, the sequence of C renders the ecPNA able toregulate the transcription and expression of the gene by repressingtranscription of the gene.

In some embodiments of the invention, A comprises a sequence selectedfrom the group consisting of the following sequences: YGRKKRRQRRR (SEQID NO: 6), GRKKRRQRRRPPQ (SEQ ID NO: 7), YARKARRQARR (SEQ ID NO: 8),YARAAARQARA (SEQ ID NO: 9), YARAARRAARR (SEQ ID NO: 10), YARAARRAARA(SEQ ID NO: 11), PKKKRKV (SEQ ID NO: 12), RQIKIWFQNRRMKWKK (SEQ ID NO:13), KKWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15),RQIKIWFPNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ ID NO: 17),RQIKIWFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRMRWRR (SEQ ID NO: 19),RRWRRWWRRWWRRWR (SEQ ID NO: 20), RQILIWFQNRRMKWKK (SEQ ID NO: 22),LLIILRRRIRKQAHAHSK (SEQ ID NO: 23), KLALKLALKALKAALKLA (SEQ ID NO: 24),and AGYLLGKINLKALAALAKKIL (SEQ ID NO: 25).

In certain embodiments of the invention, C comprises a sequence selectedfrom the group consisting of the following sequences: DFDLDMLGDFDLDMLG(SEQ ID NO: 26), MLGDFDLDMLGDFDLD (SEQ ID NO: 30), CGSDALDDFDLDML (SEQID NO: 27), PEFPGIELQELQELQALLQQ (SEQ ID NO: 28), andRHGEKWFLDDFTNNQMDQDY (SEQ ID NO: 29).

In some embodiments, C comprises a sequence selected from the groupconsisting of the sequence of an engrailed repression domain, thesequence of the HID (HDAC interaction domain) of the Sin3A protein, andMSRRKQSKPRQI (SEQ ID NO: 47).

In some embodiments of the invention there is a chemical linker betweenA and B or between B and C and the chemical linker is selected from thegroup consisting of a stable polyether, AEEA(2-aminoethoxy-2-ethoxyacetic acid), and a cleavable disulfide linkage.

In certain embodiments of the invention, the gene is a γ-globin gene.

In some embodiments, the PNA structure comprises the sequenceTACTCTAAGACTATT (PNA78: SEQ ID NO: 1).

In some embodiments, the ecPNA has the structureH₂N-CGSDALDDFDLDML-Biotin-O—B—O-YGRKKRRQRRR ((SEQ ID NO:27)-Biotin-O—B—O-(SEQ ID NO: 6)).

In other embodiments, the ecPNA has the structureH₂N-CGSDALDDFDLDML-Biotin-O-TACTCTAAGACTATT-O-YGRKKRRQRRR ((SEQ ID NO:27)-Biotin-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)).

In certain embodiments, the ecPNA has the structureBiotin-OO-DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-O-YGRKKRRQRRR(Biotin-OO-(SEQ ID NO: 26)-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)).

In some embodiments, at least one amino acid is in the form of aD-isomer.

A composition comprising the any ecPNA disclosed herein and a carrier isalso included as an embodiment of the present invention.

Certain embodiments include methods of detecting the presence of a DNAwithin the nucleus of a cell which comprises contacting the cell withthe ecPNA of claim 1 under conditions such that the ecPNA enters thecell and the PNA structure B hybridizes to the DNA to form ahybridization product, and then detecting the resulting hybridizationproduct.

Embodiments of the invention include methods for upregulatingtranscription of a γ-globin gene in a mammalian bone marrow cellcomprising contacting the cell with an ecPNA of the invention comprisedof a sequence that is capable of upregulating or activating thetranscription of a gene.

In some embodiments, the mammalian bone marrow cell is an adulterythroid cell.

Embodiments of the invention include methods for treating a β-globindisorder in a mammal comprising administering to said mammal atherapeutically effective amount of any ecPNA disclosed herein.

In some embodiments of the invention, the β-globin disorder is sicklecell anemia or β-thalassemia.

Some embodiments further comprise administering to an animal a secondagent selected from the group consisting of hydroxyurea, a short chainfatty acid (SCFA) inducer, 5-azacytidine, and a histone deacetylaseinhibitor.

In some embodiments of the invention, the short chain fatty acid (SCFA)inducer is butyrate.

In some embodiments of the invention, the histone deacetylase inhibitoris suberoylanilide hydroxamic acid (SAHA).

In some embodiments of the invention, B is from 12 to 15 nucleotides inlength.

Certain embodiments of the present invention are based on thedemonstration herein that peptide nucleic acid (PNA) molecules can beused to effectively modulate specific gene expression by conjugatingthem to two peptide sequences: (1) a cell and/or nuclear entry sequenceand (2) a transcription activation or repression domain. The embeddedchimeric peptide nucleic acid (ecPNA) molecules of the present inventionare designed to bind to a specific DNA sequence in a chromosome, therebyproviding increased specificity. The presence of cell and/or nuclearentry sequence facilitates greater efficiency of ecPNA cell and/ornuclear entry. Rather than relying on a triplex structure to passivelylead to transcriptional increase, as described in the prior art, theecPNAs of the present invention include a transcriptional activationdomain which directly activates transcription of a target gene.Furthermore, the novel ecPNAs of the present invention can include atranscriptional repression domain, which directly repressestranscription of a target gene, a function not provided by the triplexstructures of the prior art.

As demonstrated in the Examples section, below, two specific ecPNAconstructs of the invention ATF-Bio/PNA78/TAT and VP2/PNA78/TAT producedtherapeutically significant increases (7-fold and 2-fold, respectively)in γ-globin gene expression in mouse bone marrow cells engineered tocarry a yeast artificial chromosome that contains the complete humanβ-like globin locus (β-YAC). Thus, the ecPNA molecules of the inventioncan be used for the treatment of diseases, e.g., diseases or disordersassociated with β-globin defects.

Further, in certain embodiments of the invention, the ecPNA moleculescan be used to activate or repress gene expression (transcription) forthe induction of inducible pluripotent stem (iPS) cells. In a specificembodiment, ecPNA molecules are used to induce transcription of OCT4 orSOX2 or both OCT4 and SOX2 in primary human CD34+ blood cells, bonemarrow cells, neural cells and/or cord blood (CB) cells as a means togenerate iPS cells. In other embodiments, transcription of other genes,such as, but not limited to, c-MYC, KLF4, LIN28 and/or NANOG can also beactivated using ecPNA molecules of the invention. In certainembodiments, expression of any gene or combination of genes that issuitable for the induction of iPS cells is activated using a combinationof different ecPNA molecules (i.e., ecPNA molecules having differenttarget genes) of the invention. In certain embodiments, an ecPNAmolecule of the invention represses the expression (transcription) of agene, e.g., a gene associated with differentiated cells, for theinduction of iPS cells. In certain embodiments, a combination of ecPNAmolecules is administered to a cell to induce iPS cells, wherein atleast one of the ecPNA molecules in the combination activates geneexpression and at least one of the ecPNA molecules represses geneexpression, wherein the combination is suitable for the induction of iPScells. The specific combination of genes to be activated or repressedwill depend on the cell type and source

In certain embodiments, and without limitation, the invention providesthe advantage that neither viral vectors nor oncogenes are introduced,thereby providing a method with increased efficacy and safety comparedto other methods. Specifically, there is no transduction or transfectionprotocol involved, as the ecPNA molecules of the invention, while notintending to be bound by one particular theory or mechanism, are thoughtto enter the cell and/or nucleus by macropinocytosis. Mammalian cellstake up ecPNA molecules efficiently (50-95% after four hours ofincubation). In addition, effects on gene expression can be seen within24 hours. As a result, the ecPNA molecules of the invention provide auseful and efficient alternative to the present vectors and protocolsbeing used to generate iPS cells.

While not intending to be bound by a specific mechanism or theory, thePNAs within ecPNAs of the present invention are believed to function viaa single strand invasion by hybridizing with one strand of DNA of thetarget promoter sequence through Watson-Crick base pairing and replacingthe other strand of the double helix. This way the PNAs of the presentinvention avoid the limitations that follow from a triplex-formingdesign of the PNAs of the prior art (e.g., requirement for homopurine inthe target sequence and requirement for the use of base analogues, whichlimits the number of potential DNA binding sites).

By providing the novel ecPNA molecules, the present invention provides asequence-specific and efficient (i.e., low-toxic)gene-activating/repressing tool which can be used in vitro, ex vivo orin vivo to repress the expression of harmful genes (e.g., oncogenes), orto upregulate the expression of useful genes (e.g., tumor suppressorgenes or iPS cell genes), or to reactivate the expression of normallyrepressed genes (e.g., fetal-specific genes in adult cells, e.g.,embryonic and/or stem cell genes). The ecPNAs of the present inventioncan thus be used to treat various diseases treatable by transcriptionalmodulation, which include, among others, various cancers and hemoglobindisorders, as well as to generate iPS cells. For example, diseasestreatable by the molecules of the present invention include, but are notlimited to, β-thalassemia (Cooley's anemia), sickle cell disease,chronic myelogenous leukemia (CML) and other myeloproliferativediseases, and acute myeloid leukemia.

In other embodiments, the iPS cells generated using the ecPNA moleculescan be used to generate cellular models of diseases associated withsomatic gene mutations in, e.g., the bone marrow compartment or stromalcompartment. For example, iPS cells generated using the present ecPNAmolecules can be used to model acquired blood diseases and blooddisorders such as myelodysplastic syndromes, paroxysmal nocturnalhemoglobinuria, and others for which animal models are not available ordifficult to create. In addition, iPS cells carrying, e.g., aleukemia-specific cytogenetic translocation can be used to analyze howcancer stem cells develop.

In other embodiments, the iPS cell generated according to the methodsdescribed herein can be used to treat a disease or condition in asubject. For example, iPS cells can be differentiated toward a desiredcell lineage or type and administered to a patient in need of suchtreatment. As an example, and without limitation, a patient with spinalcord injury can be administered neural cells, e.g., to the site ofspinal cord injury, that were differentiated from iPS cells in vitro(e.g., from a cell line) or ex vivo (e.g. from cells obtained from thepatient or from a donor).

In other embodiments, iPS cells generated according to the methodsdescribed herein are administered to the patient without beingdifferentiated (i.e., they are administered as iPS cells).

In certain embodiments, iPS cells are preferentially induced from apatient's own somatic cells (e.g., bone marrow or peripheral bloodcells, or any other suitable cell type). While not intending to be boundby theory or mechanism, such methods provide the added advantage ofproviding therapies that are less prone to immune rejection than, forexample, therapies using embryonic stem cells, because the cells used inthe therapies described herein can be derived entirely from a patient'sown cells.

DEFINITIONS

The term “upregulate transcription” and is used to mean enhancetranscription and expression of a gene, the expression of which occurs,but at levels less than desired. The term “activate expression” is usedto mean activate a gene which otherwise being transcribed and expressed.As specified herein, the use of the ecPNAs of the present inventionleads to at least about 2-fold increase of transcription, morepreferably at least about 5-fold increase of transcription, and mostpreferably at least about 7-fold increase of transcription of a gene asmeasured by any suitable assay (e.g., quantitative mRNA analyses [e.g.,qPCR or Northern blot analysis], and/or quantitative protein analyses[e.g., Western blot or immunofluorescence analysis]).

Similarly, the term “downregulate transcription” is used interchangeablyto mean reduce the transcription and expression of a gene, theexpression of which occurs at levels greater than desired. The term“repress transcription” is used to mean eliminate transcription andexpression of a gene, the expression of which results in an undesiredeffect. As specified herein, the use of the ecPNAs of the presentinvention leads to at least about 2-fold decrease of transcription, morepreferably at least about 5-fold decrease of transcription, and mostpreferably at least about 7-fold decrease of transcription of a gene asmeasured by any suitable assay (e.g., quantitative mRNA analyses [e.g.,qPCR or Northern blot analysis], and/or quantitative protein analyses[e.g., Western blot or immunofluorescence analysis]).

As used herein, the term “detecting the presence” means marking thepresence of a DNA with an oligopeptide of a compound of the invention inany way which may be detected. In certain embodiments of the invention,an oligopeptide of a compound of the invention may be detected throughthe use of an antibody.

As used herein, the term “target gene” means the gene for which thetranscription is to be activated, upregulated, downregulated, orrepressed by a compound of the present invention.

Within the meaning of the present invention, the term“co-administration” is used to refer to administration of an ecPNA and asecond agent simultaneously in one composition, or simultaneously indifferent compositions, or sequentially within a certain time period.

As used herein, the term “inducible pluripotent stem (iPS) cell” refersto a type of pluripotent stem cell artificially derived from anon-pluripotent cell, typically an adult somatic cell, by inducing a“forced” expression of specific genes. iPS cells can also be derived bythe repression of certain genes (e.g., genes associated withdifferentiated cells) in lieu of or in addition to by activating theexpression of specific genes. The term, “induce/inducing an iPS cell”,as used herein, means that a cell having the characteristics of an iPScell (e.g., is a pluripotent stem cell) is generated. In certainembodiments, iPS cells are induced by forcing or activating theexpression of SOX2 and OCT4, and optionally, KLF4 and/or cMYC, althoughinduced expression and/or repression of other genes suitable for theinduction of iPS cells are also possible. iPS cells can be identifiedbased on the expression of one or more pluripotency markers, such as,but not limited to OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4, CRIPTO,REX1 and NANOG genes and/or proteins.

A “source cell”, as the term is used herein, refers to a cell that canbe used to obtain an iPS cell according to the methods described herein.For example, and without limitation, a human CD34+ peripheral blood cellis one source cell that can be used to induce an iPS cell, e.g., by theforced expression of OCT4 and/or SOX2.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to ±20%,preferably up to ±10%, more preferably up to ±5%, and more preferablystill up to ±1% of a given value. Alternatively, particularly withrespect to biological systems or processes, the term can mean within anorder of magnitude, preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated, the term “about” is implicit and in this context meanswithin an acceptable error range for the particular value.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition. For example, in relation to β-globin disorders, thesymptoms include anemia, tissue hypoxia, organ dysfunction, abnormalhematocrit values, ineffective erythropoiesis, abnormal reticulocytecount, abnormal iron load, splenomegaly, hepatomegaly, impairedperipheral blood flow, dyspnea, increased hemolysis, jaundice, anemiccrises and pain such as angina pectoris, etc. Within the meaning of thepresent invention, the term “treat” also denotes to arrest, delay theonset (i.e., the period prior to clinical manifestation of a disease)and/or reduce the risk of developing or worsening a disease. The term“protect” is used herein to mean prevent, delay or treat, or all, asappropriate, development or continuance or aggravation of a disease in asubject.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or pharmaceuticalcomposition that is sufficient to result in a desired activity uponadministration to an animal in need thereof. Within the context of thepresent invention, the term “therapeutically effective” refers to thatquantity of a compound or pharmaceutical composition that is sufficientto reduce or eliminate at least one symptom of a disease or disorder(e.g., a cancer or a β-globin disorder). Note that when a combination ofactive ingredients is administered the effective amount of thecombination may or may not include amounts of each ingredient that wouldhave been effective if administered individually.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to amammal (e.g., a human). Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals, and moreparticularly in humans.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. [See, e.g., Sambrook, Fritschand Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (herein“Sambrook et al, 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984);Nucleic Acid Hybridization (Hames and Higgins eds. 1985); TranscriptionAnd Translation (Hames and Higgins eds. 1984); Animal Cell Culture(Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al.eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.1994; among others.]

Embedded Chimeric Peptide Nucleic Acid Molecules of the Invention

In a most general aspect, the present invention provides an isolatedembedded chimeric peptide nucleic acid (ecPNA) molecule comprising (i) aPNA targeting a promoter region of a gene, wherein said PNA isconjugated to (ii) at least one cell or nuclear entry sequence and (iii)a transcription activation domain.

In a further general aspect, the present invention provides an ecPNAmolecule comprising (i) a PNA targeting a promoter region of a gene,wherein said PNA is conjugated to (ii) at least one cell or nuclearentry sequence and (iii) a transcription repression domain.

In a further general aspect, the present invention provides an ecPNAmolecule comprising (i) a PNA targeting a promoter region of a gene,wherein said PNA is conjugated to (ii) at least one compound whichrenders the ecPNA able to bind a receptor on a cell and (iii) atranscription repression domain.

In one specific aspect, the invention provides an ecPNA moleculecomprising (i) a PNA targeting a promoter region of a γ-globin gene,wherein said PNA is conjugated to (ii) at least one cell or nuclearentry sequence and (iii) a transcription activation domain.

In yet another embodiment, the invention provides an ecPNA consistingessentially of (i) a PNA targeting a promoter region of a γ-globin gene,wherein said PNA is conjugated to (ii) at least one cell or nuclearentry sequence and (iii) a transcription activation domain.

In yet another embodiment, the invention provides an ecPNA moleculeconsisting essentially of (i) a PNA targeting a promoter region of aSOX2 gene, wherein said PNA is conjugated to (ii) at least one cell ornuclear entry sequence and (iii) a transcription activation domain.

In another embodiment, the invention provides an ecPNA moleculeconsisting essentially of (i) a PNA targeting a promoter region of anOCT4 gene, wherein said PNA is conjugated to (ii) at least one cell ornuclear entry sequence and (iii) a transcription activation domain.

In still another embodiment, the invention provides an ecPNA moleculeconsisting essentially of (i) a PNA targeting a promoter region of aKLF4 gene, wherein said PNA is conjugated to (ii) at least one cell ornuclear entry sequence and (iii) a transcription activation domain.

In yet another embodiment, the invention provides an ecPNA moleculeconsisting essentially of (i) a PNA targeting a promoter region of anyone or more genes selected from c-MYC, LIN28, NANOG, PRDM14, and NFRKB,wherein said PNA is conjugated to (ii) at least one cell or nuclearentry sequence and (iii) a transcription activation domain.

Any PNA sequence which corresponds to a sequence comprised in thepromoter sequence of a target gene can be used within the ecPNAmolecules of the invention. In preferred embodiments, the PNA sequencesare complementary to a sequence within the 200 base pair (bp) regionthat is upstream of the transcription start site of a target gene, asshown, e.g., in FIGS. 7-10 and 15-18. While not intending to be bound bytheory, this 200 bp region is important for transcriptional regulationof gene expression.

In other embodiments, the PNA sequence is complementary to a sequencewithin the 400, 350, 300, or 250 base pair (bp) region that is upstreamof the transcription start site of a target gene. In some embodiments,the PNA sequence can comprise a nucleic acid sequence that iscomplementary to a sequence that is downstream of the transcriptionstart site of a target gene.

In certain embodiments, the PNA sequence can comprise one or morenucleic acid substitutions. For example, a nucleic acid residue can besubstituted, e.g., with a different nucleic acid, for example, andwithout limitation, inosine, pseduoisocytosine, 2-thiouracil, and/ordiaminopurine. PNA sequences containing such substitutions preferablyalthough not necessarily retain similar activity (i.e., ability toactivate or repress transcription of a target gene), compared to the PNAmolecule without the one or nucleic acid substitutions. In certainembodiments, a PNA molecule comprises a sequence that is at least 70%,75%, 80%, 85%, 90%, or 95% identical to a sequence that is complementaryto a sequence within the 200 base pair (bp) region that is upstream ofthe transcription start site of a target gene.

Thus, in certain embodiments, a PNA sequence that targets human OCT4will have a nucleic acid sequence of about 12 nucleotides (nt) to about15 nt that is complementary to a sequence comprised in the followingnucleic acid sequence:CCTGCACTGAGGTCCTGGAGGGGCGCCAGTTGTGTCTCCCGGTTTTCCCCTTCCACAGACACCATTGCCACCACCATTAGGCAAACATCCTTCGCCTCAGTTTCTCCCCCCACCTCCCTCTCCTCCACCCATCCAGGGGGCGGGGCCAGAGGTCAAGGCTAGTGGGTGGACTGGGGAGGGAGGGAGAGAGGGGTTGAGTAGTC (SEQ ID NO: 56) (FIG. 7).

In certain embodiments, a PNA sequence that targets human SOX2 will havea nucleic acid sequence of about 12 nt to about 15 nt that iscomplementary to a sequence comprised in the following nucleic acidsequence: GCTCTGGGAGCCTCCTCCCCCTCGCCTGCCCCCTCCTCCCCCGGCCTCCCCCGCGCGGCCGGCGGCGCGGGAGGCCCCGCCCCCTTICATGCAAAACCCGGCAGCGAGGCTGGGCTCGAGTGAGGAGCCGCCGCGCGCTGATTGOTCGCTAGAAACCCATATTCCCTGACAGCCCCCGTCACATGGATGGTTGT (SEQ ID NO: 57) (FIG. 8).

In certain embodiments, a PNA sequence that targets human KLF4 will havea nucleic acid sequence of about 12 nt to about 15 nt that iscomplementary to a sequence comprised in the following nucleic acidsequence: CCCAGCCCCGCCCGCGCCCCTCCTTCCCCTCCCCCGCCCCCACGTGCGCCGAGTTTGTTGATTTAGCTGCCATAGCAACGATGGAAGGGAGCCTCGGGGGGGGCGGAGAGAAGAAAGGGAGGGGCGGGGCATGGGAGAAGGCGGAGGAAAAGGCTGTAGCGAAGGAAGTTATAAGTAAGGAACGCGCGCCGGCGGCCGGC (SEQ ID NO: 58) (FIG. 9).

In certain embodiments, a PNA sequence that targets human c-MYC willhave a nucleic acid sequence of about 12 nt to about 15 at that iscomplementary to a sequence comprised in the following nucleic acidsequence: TTACTCT GTTTACATCCTAGAGCTAGAGTGCTCGGCTGCCCGGCTGAGTCTCCTCCCCACCTTCCCCACCCTCCCCACCCTCCCCATAAGCGCCCCTCCCGGGTTCCCAAAGCAGAGGGCGTGGGGGAAAAGAAAAAAGATCCTCTCTCGCTAATCTCCGCCCACCGGCCCTTrTATAATGCGAGGGTCTGGACGGCTGAG (SEQ ID NO: 59) (FIG. 10).

In certain embodiments, a PNA sequence that targets human LIN28 willhave a nucleic acid sequence of about 12 nt to about 15 nt that iscomplementary to a sequence comprised in the following nucleic acidsequence: TCAAA CCTCAAGGTTCTGAGAAGGGACACCCCAGAGGTGTCAGAGACCGGAGTIGTGGGGGAGGGCCGGAGCTGGAGCCGGAGGGAAAGGGAGGGGAAAGGAGAGGGAGGGGAGGGGAGGGGGCTGCCCGCGGGGGGTTGGGTCATTGTCTTTTAGAATTTGGGAGCCTITGAAAAGCCGTGGGCCCTCCCACCGCTATT (SEQ ID NO: 60) (FIG. 15).

In certain embodiments, a PNA sequence that targets human NANOG willhave a nucleic acid sequence of about 12 nt to about 15 nt that iscomplementary to a sequence comprised in the following nucleic acidsequence: CCGACTCCGCACGCCTGGAGCGGCAATACTGCCTGCCCTAGAAGGCCAGCGGCGAGTGCTCGCCACTAGGGTCCCAGGGAGAGTTTGGAAAACTGATGAGTTAAGTGAGCGACCCCAGGGGGACAGAGGGCGAGTCGAGAGTCGGCCAATGGCTGCGGTGGGCGGGGAGAAGACGACGCGGGGATCTGCGTGGGCCGGGTC (SEQ ID NO: 61) (FIG. 16).

In certain embodiments, a PNA sequence that targets human PRDM14 willhave a nucleic acid sequence of about 12 nt to about 15 nt that iscomplementary to a sequence comprised in the following nucleic acidsequence: AGTATTTGTTGCTGGGTTTGTCTTCAGGTTCTGTTGCTCGGTTTTCTAGTTCCCCACCTAGTCTGGGTTACTCTGCAGCTACTTTTGCATTACAATGGCCTTGGTGAGACTGGTAGACGGGATTAACTGAGAATTCACAAGGGTGGGTCAGTAGGGGGTGTGCCCGCCAGGAGGGGTGGGTCTAAGGTGATAGAGCCTTC (SEQ ID NO: 62) (FIG. 17).

In certain embodiments, a PNA sequence that targets human NFRKB willhave a nucleic acid sequence of about 12 nt to about 15 nt that iscomplementary to a sequence comprised in the following nucleic acidsequence: TCTGGGGCCTCTGGAAGCTCCTAAATCAGGTGAGACGCGCAAGCAGGCTGGAACCTGCATCCCCAGGCCCAGCCGCGCGCAGGCACCCTCGCCGACCCTCCGCTCTCCCGAGCCGCTCCAGGACCCGCCCGCTGTGGCCCCGCCCCGGCACCCTCCAGGCCCCGCCCCGCGCTGCCCCGCCCCTTCCGCCGCGCAGGCCC (SEQ ID NO: 63) (FIG. 18).

In some embodiments the PNA structure is from 12 nt to 15 nt in length,however, shorter or longer sequences may be possible. In certainembodiments, the PNA structure will be between 12 and 30 nucleotides inlength, e.g. between 12 and 25 nucleotides, often between 12 and 30nucleotides in length.

In some embodiments, the PNA of the ecPNA molecule comprises thesequence TACTCTAAGACTATT (PNA78) (SEQ ID NO: 1).

The sequence of the PNA structure may be capable of hybridizing with aDNA within the nucleus of a cell. In some embodiments, the sequence ofthe PNA structure will be may be complementary to a sequence within thepromoter region of a gene. Non-limiting examples of gene promoters withrespect to which complementary PNA sequences have been described arelisted in Table 1. The references describing these PNAs are also citedin Table 1 and are hereby incorporated by reference into the presentdisclosure. In additional embodiments, the sequence of the PNA structuremay be complementary to a sequence within a promoter region bound by apolymerase, such as T7, SP6, or T3 RNA polymerase. [See also Hamilton etal., Specific and nonspecific inhibition of transcription by DNA, PNA,and phosphorothioate promoter analog duplexes. Bioorganic & MedicinalChemistry Letters 6(23):2897-2900 (1996); and Larsen and Nielsen,Transcription-mediated binding of peptide nucleic acid (PNA) todouble-stranded DNA: sequence-specific suicide transcription. NucleicAcids Res. 24:458-463 (1996).]

TABLE 1 Genes with Promoters for which PNAs have been Targeted GenePNA Sequence(s) Reference BCL2 GGGCGGAGGOnyshchenko et al., Stabilization GCCAGGGAof G-quadruplex in BCL2 promoter region in double-stranded DNA byinvading short PNAs. Nucleic Acids Res.37(22):7570-80 (2009) PRαCTTTCTCCTCCCTCT Kaihatsu, K., Janowski, B.A. & Corey, (SEQ ID NO. 80)D.R., Recognition of chromosomal DNA CCTCCCCCby PNAs. Chem Biol II, 749-758 (2004) CCTTTTCCCTCCTCCCT (SEQ ID NO. 81)PRβ TGTCTGGCCAGTCCACAGC Hu and Corey, Inhibiting gene (SEQ ID NO. 82)expression with peptide nucleic acid TGTCTGGCCAGTC(PNA)- peptide conjugates that target (SEQ ID NO. 83)chromosomal DNA. Biochemistry 46(25): TGTCTGGCCAGTCCA 7581-9 (2007)(SEQ ID NO. 84) HER-2 CTCCTCCTC Stankova et al., Mechanism of PNACCTCCTCCT transport to the nuclear compartment.Ann NY Acad Sci. 1082:27-30 (2006) IL-2Rα TCTCCCTCTCCTTTTVickers et al., Inhibition of NF-B (SEQ ID NO. 85)specific transcriptional activation TTTTCCTCTCCCTby PNA strand invasion. Nucleic Acids (SEQ ID NO. 86)Research 23(15):2003-8 (1995) lacUV5 TTTTTTCTTTTMollegaard et al., Peptide nucleic (SEQ ID NO. 87)acid. DNA strand displacement loopsas artificial transcription promoters. PNAS 91(9):3892-5 (1994)

Any oligopeptide may be used as a component of the compound of theinvention since in principle any oligopeptide can be detectable. Theoligopeptide structures of the compounds of this invention may be orinclude epitope tags such as V5-tag, Myc-tag, HA-tag, FLAG-tag, GST-tag,and His-tags or any other amino acid sequence for which antibodies withsuitable specificity and affinity are generated. [See also Huang andHonda, CED: a conformational epitope database. BMC Immunology 7:7http://www.biomedcentral.com/1471-2172/7/7#B1. Retrieved Feb. 16, 2011(2006); and Walker and Rapley, Molecular biomethods handbook. Pg. 467(Humana Press, 2008).] One of ordinary skill in the art will understandessentially any oligopeptide may be used as a marker in the inventionand thus be a component of the compounds of the present invention.

In general the oligopeptide of the invention will range from 5 to 50amino acids in length, most typically from 5 to 30 amino acids inlength, e.g. from 10-30 or from 12-25 amino acids in length.

Those skilled in the art will be aware of how to produce antibodymolecules when provided with an oligonucleotide of the presentinvention. For example, polyclonal antisera or monoclonal antibodies canbe made using standard methods. A mammal, (e.g., a mouse, hamster, orrabbit) can be immunized with an immunogenic form of the oligonpeptidewhich elicits an antibody response in the mammal. Techniques forconferring immunogenicity on a oligopeptide include conjugation tocarriers or other techniques well known in the art. For example, theoligopeptide can be administered in the presence of an adjuvant. Theprogress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassay can beused with the immunogen as antigen to assess the levels of antibodies.Following immunization, antisera can be obtained, and, if desired IgGmolecules corresponding to the polyclonal antibodies may be isolatedfrom the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art. Hybridoma cells can be screened immunochemically for productionof antibodies which are specifically reactive with the oligopeptide, andmonoclonal antibodies isolated.

To enhance their immunogenicity, it is well-known to conjugate smalloligopeptide fragments to a hapten, such as, for example, dinitrophenyl(DNP), m-maleimidobenzoyl-N-hydroxyl-N-hybroxysuccinimide ester (MBS),or m-amino benzene sulphonate. A “hapten” is a non-immunogenic moleculethat will react with a preformed antibody induced by an antigen orcarrier molecule. Alternatively, the immunogenicity of oligopeptides maybe enhanced by conjugating the oligopeptide to a carrier molecule, suchas, for example, an antigenic oligopeptide, that may be conjugated to ahapten. As will be known to those skilled in the art, a “carrier” isgenerally an antigenic molecule. Preferred carrier molecules for thispurpose include ovalbumin, KLH, and PHA.

The term “antibody” as used herein, is intended to include fragmentsthereof which are also specifically reactive to an oligopeptide asdescribed herein.

Immunoassays are useful in detecting the presence of an oligopeptide asdisclosed herein, in a cell. Such an immunoassay is of particular use indetecting the presence of a DNA in a cell in certain embodiments of theinvention. Immunoassays are also useful for the quantitation of said DNAin a cell. The invention described herein extends to all such uses ofimmunointeractive molecules and diagnostic assays requiring saidimmunoassays for their performance.

A wide range of immunoassay techniques may be such as those described inU.S. Pat. Nos. 4,016,043, 4,424, 279 and 4,018,653. These methods may beemployed for detecting the presence of a DNA in some embodiments theinvention.

Any cell or nuclear entry sequence can be used within the ecPNAmolecules of the invention. In a preferred embodiment, the cell ornuclear entry sequence comprises at least one sequence selected from thegroup consisting of: (a) a TAT peptide having the sequence YGRKKRRQRRR(SEQ ID NO: 6), (b) a TAT peptide variant having the sequence selectedfrom GRKKRRQRRRPPQ (SEQ ID NO: 7), YARKARRQARR (SEQ ID NO: 8),YARAAARQARA (SEQ ID NO: 9), YARAARRAARR (SEQ ID NO: 10), and YARAARRAARA(SEQ ID NO: 11), (c) an NLS peptide having the sequence PKKKRKV (SEQ IDNO: 12), (d) a penetratin peptide having the sequence selected fromRQIKIWFQNRRMKWKK (SEQ ID NO: 13), KKWKMRRNQFWIKIQR (SEQ ID NO: 14),RQIKIWFQNRRMKWKK (SEQ ID NO: 15), RQIKIWFPNRRMKWKK (SEQ ID NO: 16),RQPKIWFPNRRMPWKK (SEQ ID NO: 17), RQIKIWFQNMRRKWKK (SEQ ID NO: 18),RQIRIWFQNRRMRWRR (SEQ ID NO: 19), and RRWRRWWRRWWRRWR (SEQ ID NO: 20),(e) an Antennapedia domain pAntp(43-58) having the sequenceRQILIWFQNRRMKWKK (SEQ ID NO: 22), (f) pVEC(615-632) having the sequenceLLIILRRRIRKQAHAHSK (SEQ ID NO: 23), (g) a model amphipathic peptide(MAP) having the sequence KLALKLALKALKAALKLA (SEQ ID NO: 24); and (h) atransportan 10 peptide having the sequence AGYLLGKINLKALAALAKKIL (SEQ IDNO: 25).

Any transcription activation or repression domain can be used within theecPNAs of the invention. In a preferred embodiment, the transcriptionactivation domain comprises at least one sequence selected from thegroup consisting of: (a) a VP2 domain having the sequenceDFDLDMLGDFDLDMLG (SEQ ID NO: 26) or the sequence MLGDFDLDMLGDFDLD (SEQID NO: 30), (b) an ATF-14 domain having the sequence CGSDALDDFDLDML (SEQID NO: 27), (c) an AH domain having the sequence PEFPGIELQELQELQALLQQ(SEQ ID NO: 28), and (d) a Gal80BP domain having the sequenceRHGEKWFLDDFTNNQMDQDY (SEQ ID NO: 29). Preferred repression domainsinclude the engrailed repression domain, the HID (HDAC interactiondomain) from the Sin3A protein, and MSRRKQSKPRQI (SEQ ID NO: 47).MSRRKQSKPRQI is a repression motif shared by the transcriptionalrepression proteins FOG12 and Sall1. [See, Manwani and Bieker, Exp. Hem.35:39-47 (2007); Lin et al., The N termini of Friend of GATA (FOG)proteins define a novel transcriptional repression motif and asuperfamily of transcriptional repressors. J Biol Chem 279(53):55017-23(2004); and Lauberth and Rauchman, A conserved 12-amino acid motif inSAll1 recruits the nucleosome remodeling and deacetylase corepressorcomplex. J Biol Chem 281(33)23922-31 (2006)]

Aspects of the invention relate to ecPNAs that downregulate thetranscription of specific target genes. ecPNAs which downregulate thetranscription of specific target genes may comprise the FOG12 repressionmotif, which has amino acids in the sequence: MSRRKQSKPRQI (SEQ ID NO:47). When this motif is fused with the TAT-PNA78 sequence, the resultantTAT-PNA78-MSRRKQSKPRQI (referred to herein as “TAT-PNA78-FOG12”)(TAT-PNA78-(SEQ ID NO: 47)) molecule represses, rather than activates, γglobin expression.

In one specific embodiment, the ecPNA molecule is ATF-Bio/PNA/TAT havingthe structure H₂N-CGSDALDDFDLDML-Biotin-O-PNA-O-YGRKKRRQRRR ((SEQ ID NO:27)-Biotin-O-PNA-O-(SEQ ID NO: 6)). In another specific embodiment, theecPNA molecule is ATF-Bio/PNA78/TAT having the structureH₂N-CGSDALDDFDLDML-Biotin-O-TACTCTAAGACTATT-O-YGRKKRRQRRR ((SEQ ID NO:27)-Biotin-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)). In yet another specificembodiment, the ecPNA molecule is VP2/PNA78/TAT having the structureBiotin-OO-DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-O-YGRKKRRQRRR(Biotin-OO-(SEQ ID NO: 26)-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)). In theabove structures, “O” represents the stable polyether linker, AEEA(2-aminoethoxy-2-ethoxyacetic acid). In a preferred embodiment, all TATamino acids in such ecPNA molecules are in D-isomer form. In any ofthese embodiments, the inclusion of Biotin (Bio) is optional. Othermarkers useful for visualizing an ecPNA molecule, such as but notlimited to GFP and other fluorescent markers, may also be optionallyincluded in the ecPNA molecules.

In certain embodiments, the ecPNA molecule has TAT in the D or L form.In certain embodiments, the transactivating domain is in the D or Lform.

Methods for producing PNA-peptide conjugates of the present inventionare well known in the art and include, among others, solid-phasesynthesis and fragment ligation [see, e.g., de Koning et al., CurrentOpinion in Chemical Biology, 2003, 7(6):734-740]. Linkers useful in thePNA-peptide conjugates of the present invention include, for example,stable polyether, AEEA (2-aminoethoxy-2-ethoxyacetic acid), cleavabledisulfide linkages, or other linkers that are generally known in theart.

In some embodiments, an ecPNA molecule is used to activate γ-globinexpression in a patient by ex vivo treatment of bone marrow cells priorto placement of the bone marrow cells back into the sickle orthalassemic patient.

Aspects of the invention relate to the systemic injection of ecPNAs. Insome embodiments, the ecPNA comprises a compound that binds a receptoron the surface of a cell. In some embodiments, the ecPNA comprises acompound that is capable of binding to a receptor that is present on thesurface of a certain type or types of cells. Thus, embodiments of theinvention encompass the specific targeting of a specific cell type ortypes within a subject with an ecPNA, wherein the ecPNA that isadministered to the subject comprises a compound that binds to areceptor on the specific cell type(s). For instance, erythroid cellscould be targeted by using an ecPNA which comprises a compound thatbinds to the erythropoietin (Epo) receptor. The compound may be an Epomimetic. Epo mimetics and other compounds that bind the Epo receptorinclude but are not limited to Epo mimetic peptides (EMPs), Hematide,and others discussed in Bunn H F. New agents that stimulateerythropoiesis. Blood. February 1; 109(3):868-73 (2007), the entirecontents of which are hereby incorporated herein by reference. In anon-limiting example, an EMP-PNA78-ATF ecPNA which upregulates γ-globinexpression is administered to a subject systemically, but entersspecifically into erythroid cells in the subject which has been injectedwith the ecPNA after binding of the EMP portion of the ecPNA to theerythropoietin receptor on those cells (see, e.g. FIG. 21).

Compositions

While it is possible to use a composition provided by the presentinvention for therapy as is, it may be preferable to administer it in apharmaceutical formulation, e.g., in admixture with a suitablepharmaceutical excipient, diluent, or carrier selected with regard tothe intended route of administration and standard pharmaceuticalpractice.

Accordingly, in one aspect, the present invention provides apharmaceutical composition or formulation comprising at least one ecPNAmolecule of the invention, or a pharmaceutically acceptable derivativethereof, in association with a pharmaceutically acceptable excipient,diluent, and/or carrier. The excipient, diluent and/or carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.Preferably, the at least one ecPNA molecule is present in suchcompositions in a therapeutically effective amount.

The compositions of the invention can be formulated for administrationin any convenient way for use in human or veterinary medicine.

Carrier

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. In some embodiments, carriers arepharmaceutical carriers. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Alternatively, thecarrier can be a solid dosage form carrier, including but not limited toone or more of a binder (for compressed pills), a glidant, anencapsulating agent, a flavorant, and a colorant. Suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin (1990, Mack Publishing Co., Easton, Pa.18042).

Formulations

The compositions and formulations of the present invention may comprisepharmaceutically or otherwise acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80. Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hylauronic acid may also be used. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435 1712 which are herein incorporated byreference.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants, preserving, wetting,emulsifying, and dispersing agents. The pharmaceutical compositions maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured using sterile water, or some other sterileinjectable medium, immediately before use.

Methods of the Invention

In conjunction with ecPNA molecules, the present invention providesmethods for using such molecules, or compositions containing suchmolecules, to activate or repress transcription of a target gene.Important genes that may be targeted by the ecPNA molecules of thepresent invention include, for example, activation of γ-globin (fortreatment of β-thalassemia or sickle cell disease, activation of the p15tumor suppressor gene for treatment of cancer, and repression of theBCR/ABL oncogene (for treatment of leukemia).

In one specific embodiment, the present invention provides a method forupregulating transcription of a γ-globin gene in a bone marrow mammaliancell comprising contacting said cell with an ecPNA molecule having thestructure H₂N-CGSDALDDFDLDML-Biotin-O-TACTCTAAGACTATT-O-YGRKKRRQRRR(ATF-Bio/PNA78/TAT) ((SEQ ID NO: 27)-Biotin-O-(SEQ ID NO: 1)-O-(SEQ IDNO: 6)). In another specific embodiment, the present invention providesa method for upregulating transcription of a γ-globin gene in a bonemarrow mammalian cell comprising contacting said cell with an ecPNAmolecule having the structureBiotin-OO-DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-O-YGRKKRRQRRR (VP2PNA78/TAT) (Biotin-OO-(SEQ ID NO: 26)-O-(SEQ ID NO: 1)-O-(SEQ ID NO:6)).

Since the PNA portion of the ecPNA molecules of the present inventioncan be directed to any promoter region of a target gene of interest, thepresent invention provides a potentially powerful yet low-toxictreatment for any disorder that can be alleviated by activation orrepression of a target gene.

In certain specific embodiments, the invention provides a method fortreating a β-globin disorder in a mammal comprising administering to themammal a therapeutically effective amount of an ecPNA molecule whichupregulates the transcription of a γ-globin gene. Non-limiting examplesof encompassed β-globin disorders include sickle cell anemia andβ-thalassemia.

The human γ-globin (HBG1) mRNA sequence has GenBank Accesion No.NM_(—)000559 (SEQ ID NO: 42). The human γ-globin amino acid sequence hasGenBank Accession No. NP_(—)000550 (SEQ ID NO: 43). The human β-globinmRNA sequence has GenBank Accesion No. NM_(—)000518 (SEQ ID NO: 44). Thehuman 3-globin amino acid sequence has GenBank Accession No.NP_(—)000509 (SEQ ID NO: 45). The gene region on chromosome 11containing the human epsilon, gamma G, gamma A, beta 1 pseudogene,delta, and beta-3′ globin genes has GenBank Accession No. NG_(—)000007(SEQ ID NO: 46).

Induction of iPS Cells

In conjunction with the ecPNAs of the invention, methods for inducingiPS cells comprising administering ecPNA molecules to a cell areprovided.

Induced pluripotent stem (iPS) cells may be used to generate anydifferentiated tissue from an individual in need of the differentiatedtissue. The differentiated tissue may then be provided to theindividual, avoiding tissue rejection and other complications that mayarise from an individual receiving differentiated tissue from a non-selfsource. Blood cells may be induced to iPS status by the introduction ofonly two transcription factors (OCT4 and SOX2), and ecPNA moleculesdescribed herein are a means to efficiently and selectively activate theOCT4 and SOX2 genes in vivo. ecPNA molecules are also described in PCTInternational Application Publication No. WO2011/103215, and (Chen etal. Design of Embedded Chimeric Peptide Nucleic Acids that EfficientlyEnter and Accurately Reactivate Gene Expression In Vivo. PNAS 107,16846-16851 (2010)), the entire contents of which are herebyincorporated herein by reference. In some embodiments, ecPNA moleculesthat activate OCT4 and SOX2 in primary human blood cells are used as ameans to induce them to iPS status.

In certain embodiments, the iPS cells are generated in vitro.Preferably, iPS cells are induced by introducing an ecPNA molecule thatupregulates transcription of OCT4. In certain embodiments, iPS cells areinduced by introducing an ecPNA molecule that upregulates transcriptionof OCT4 and an ecPNA molecule that upregulates transcription of OCT4. Incertain embodiments, ecPNA molecules specific for SOX2 and/or OCT4 areadministered to a cell with additional ecPNA molecules specific for theinduction of one or more other genes. For example, the one or more othergenes can include without limitation c-MYC, KLF4, LIN28, NANOG, PRDM14and NFRKB [see, Chia, N Y et al. Nature; 2010; 468(7321)316-320]. TheecPNA molecules can target any gene useful for the induction of iPScells, and can be used in any combination (of different target genespecificities) suitable for the induction of iPS cells.

Any suitable cell type can be used for the generation of iPS cells(these cells are referred to herein as “source cells”). In a preferredembodiment, iPS cells are induced from CD34+ peripheral blood cells,bone marrow cells, and/or CD133+ cord blood (CB) cells. Other suitablesource cells include without limitation fibroblasts, hepatocytes,gastric epithelial cells, mesenchymal cells, neural cells [see, Kim, J.B. et al., Nature. 2009 Oct. 1; 461(7264):649-3] and other somatic cells[see, Aoi et al, (2008) Science 321: 699-702; Park et al, 2008; Nature451: 141-146; Tsai et al., Stem Cells; 28, 221-228 (2010); Szabo et al.Nature, in press, doi:10.1038/nature09591]. The source cells for iPSinduction can be derived from any suitable mammalian source cell, suchas but not limited to murine or primate cells, and preferably humancells. The source cells may be obtained, for example and withoutlimitation, from a cell line, from a patient or from a donor source.Methods for the isolation and culture of source cells are known in theart. The following methods are provided as examples and are notlimiting. Any suitable method for the isolation of source cells for iPScell generation and for their culture, are contemplated for use in thepresent invention.

For the isolation and culture of peripheral blood cells, the followingprocedure can be followed. Mobilized peripheral blood can be obtained asfollows: the donor is injected with 5 μg/kg/day for 3 days with Neupogen(G-CSF) manufactured by Amgen. On the fourth day, apheresis of 7 bloodvolumes is performed on a Cobe Apheresis machine, and 300 ml of blood iscollected. Using this method, the yield of CD34+ cells is about 1%. Forculture, mobilized peripheral blood CD34+ cells (Allcells, mPB014F) canbe maintained in IMDM (Invitrogen) containing 15% fetal bovine serum(StemCell Technologies) supplemented with hSCF (100 ng/ml), hFlt3L (100ng/ml), and Interleukin-3 (20 ng/ml) (Peprotech). CD34+ cells grown inculture for four days can then be used for treatment with ecPNAmolecules of the invention. Once iPS cells begin to form, cell clusterscan be mechanically scraped into strips and transferred to 6-well, lowattachment plates in differentiation medium consisting of knockout DMEM(Invitrogen) supplemented with 20% fetal bovine serum (StemCellTechnologies), 0.1 mM non-essential amino acids (Invitrogen), 1 mML-glutamine (Invitrogen), 50 μg/ml ascorbic acid (Sigma), and 2 mg/mlhuman holo-transferrin (Sigma) and 0.1 mM β-mercaptoethanol (Sigma).[See, Loh et al. (2009) Blood; 113(22):5476-5479.]

For the isolation of CB cells, the following procedure can be followed.Umbilical CB samples can be obtained from a suitable source, such ascord blood bank. Mononuclear cells (MNC) are isolated from CB usingLympholyte-H (Cederlane, Ontario, CA) density gradient centrifugation.CD133+ cells are positively selected using Mini-Macs immunomagneticseparation system (Miltenyi Biotec, Bergisch Gladbach, Germany).Purification efficiency is verified by flow cytometric analysis stainingwith CD133-phycoerythrin (PE; Miltenyi Biotec, Bergisch Gladbach,Germany) antibody.

For the culture of CB cells and induction of iPS cells, CB CD133+ cells(0.08×105 cells per ml) are pre-stimulated for 24 h in DMEM supplementedwith 10% of FBS in the presence of SCF (50 ng/ml) +Flt3 (50 ng/ml) +TPO(10 ng/ml) +IL-6 (10 ng/ml)(PeproTech). Multi-well non-tissueculture-treated plates were coated with retronectin (Takara, Otsu,Japan, www.takara-bio.com), a fibronectin fragment CH-296 (15 mg/cm2),and preloaded by centrifuging the plates with a filtered equal molar mixof ecPNA molecules specific for target genes (e.g., OCT4 and/or SOX2,and optionally, KLF4, c-MYC, LIN28, NANOG, PRDM14, and/or NFRKB) at2,500 RPM for 30 minutes. About 80,000 CD133+ cells are plated in thepresence of DMEM+10% FBS and the cytokine cocktail mentioned above.[see, Giorgetti, A. et al. (2009) Cell Stem Cell; 5:353-357.]

For the analysis of gene expression in iPS cells, any suitable method,such as PCR or Southern blot can be used. For example, genomic DNA fromthe iPS cells can be isolated using All Prep DNA/RNA columns (Qiagen),following manufacturer's guidelines. 4 □g of genomic DNA digested with40 U of either PstI or HindIII restriction enzyme (New England Biolabs)can be loaded and electrophoresed on a 1% agarose gel, transferred to aneutral nylon membranes (Hybond-N, Amersham) and hybridized withDIG-dUTP labeled probes generated by PCR using the PCR DIG ProbeSynthesis Kit (Roche Diagnostics). Probes can be detected by anAPconjugated DIG-Antibody (Roche Diagnostics) using CDP-Star(Sigma-Aldrich) as a substrate for chemiluminescence. Conditions are asper the instructions of the manufacturer. The probes can be generatedusing SOX2, OCT4, KLF4 and c-MYC cDNAs as templates with the followingprimers (F, forward; R, reverse): SOX2 F 5′-AGTACAACTCCATGACCAGC-3′ (SEQID NO: 48); SOX2 R 5′-TCACATGTGTGAGAGGGGC-3′ (SEQ ID NO: 49): OCT4 F5′-TAAGCTTCCAAGGCCCTCC-3′ (SEQ ID NO: 50); OCT4 R5′-CTCCTCCGGGTTITGCTCC-3′ (SEQ ID NO: 51); KLF4 F5′-AATTACCCATCCTTCCTGCC-3′ (SEQ ID NO: 52); KLF4 R5′-TTAAAAATGCCTCTTCATGTGTA-3′ (SEQ ID NO: 53); c-MYC F5′-TCCACTCGGAAGGACTATCC-3′ (SEQ ID NO: 54); c-MYC R5′-TTACGCACAAGAGTTCCGTAG-3′ (SEQ ID NO: 55). Probes and primers can bereadily designed for any target gene encompassed by the invention by oneof ordinary skill in the art.

Following iPS cell induction according to the methods described hereinor other suitable methods, iPS cells can be analyzed for expression ofmarkers characteristic of iPS cells in order to confirm successful iPScell induction. Examples of suitable markers include OCT4, SOX2,TRA-1-81, TRA-1-60, SSEA3, SSEA4, CRIPTO, REX1 and NANOG genes and/orproteins, although a number of other markers are also possible. Incertain embodiments, immunofluorescence can be used to characterize iPScells. iPS can be grown on plastic coverslide chambers and fixed with 4%paraformaldehyde (PFA). The following antibodies can be ud for staining:TRA-1-60 (MAB4360, 1:200), TRA-1-81 (MAB4381, 1:200), SOX2 (AB5603,1:500) all Chemicon, SSEA-4 (MC-813-70, 1:2), SSEA-3 (MC-631, 1:2) allIowa, Tuj1 (1:500; Covance) α-fetoprotein (1:400; Dako), α-actinin(1:100; Sigma), OCT4 (C-10, SantaCruz, sc-5279, 1:100), NANOG (EverestBiotech EB06860, 1:100), GATA 4 (1:50, SantaCruz), smooth muscle actin(1:400, Sigma), FoxA2 (1:50 R&D System), GFAP (1:1000, Dako),α-sarcomeric actin (1:400, Sigma), Anti-Flag (Sigma M2). Images can betaken using, e.g., a Leica SP5 confocal microscope. Direct AP activitycan be analyzed using an Alkaline Phosphatase Blue/Red Membranesubstrate solution kit (Sigma) according to the manufacturer'sguidelines. Additional methods for the culture and characterization ofiPS cells are described in detail in Giorgetti, A. et al. (2009), supra;Loh et al. (2009) supra; and Takahashi, K. et al. (2007) supra.

In certain embodiments, iPS cells generated according to methods of theinvention can be then be differentiated into specific cell types invitro. For example, once iPS cells are generated, it may then bedesirable to generate a specific cell type from these cells, e.g., fortreatment of a patient in need of treatment with a specific type ofcell. Methods for differentiating iPS cells are known in the art [see,Giorgetti, A. et al. (2009), supra.; and Warren, et al. (2010) Stem Cell(7) in press; PMID: 20888316].

Dosage and Administration

ecPNA-containing compositions of the invention can be directly orindirectly administered to a subject (e.g. patient). Indirectadministration is performed, for example, by administering thecomposition to cells (e.g., bone marrow cells, peripheral blood cells,CB cells or any other suitable cell) in vitro or ex vivo (i.e. thesource cells may be derived from cell lines (in vitro) or obtained fromthe patient to be treated or from another donor source (ex vivo)), andsubsequently administering the treated cells to the patient.) ex vivoand subsequently introducing the treated cells to the patient. The cellsmay be obtained from the patient to be treated or from a geneticallyrelated or unrelated patient. Related patients offer some advantage bylowering the immunogenic response to the cells to be introduced. Forexample, using techniques of antigen matching, immunologicallycompatible donors can be identified and utilized. Following treatment ofthe cells with a composition of the invention, the cells may beadministered to a patient in need of such treatment by any suitableroute, such as oral, parenteral, sublingual, rectal such as suppositoryor enteral administration, or by pulmonary absorption or topicalapplication. In a preferred embodiment, the cells are administeredintravenously.

Direct administration of an ecPNA-containing composition to a subjectmay also be by oral, parenteral, sublingual, rectal such as suppositoryor enteral administration, or by pulmonary absorption or topicalapplication.

In certain embodiments iPS cells generated according to the methodsdescribed herein are administered to a patient. In some embodiments, theiPS cells are administered intravenously, although it is possible toadminister the cells subcutaneously, intranasasally, orally, or by anyother suitable route of administration. Methods for administering cellsto a subject are known in the art.

In certain embodiments, an ecPNA molecule-containing composition or aniPS cell generated according to the methods described herein, or a celldifferentiated from an iPS cell generated according to the methodsdescribed herein is administered directly to a site of injury or diseasein a subject. For example it may be useful to directly administer anecPNA molecule for inducing an iPS cell directly to a site where itwould be useful to induce stem cell development. Such stem cells couldthen differentiate into a desired tissue type under the control of thecytokine milieu present at the targeted site.

Parenteral administration may be by intravenous injection, subcutaneousinjection, intramuscular injection, intra-arterial injection,intrathecal injection, intraperitoneal injection or direct injection orother administration to one or more specific sites. Injectable forms ofadministration are sometimes preferred for maximal effect in, forexample, bone marrow. When long-term administration by injection isnecessary, venous access devices such as medi-ports, in-dwellingcatheters, or automatic pumping mechanisms are also preferred whereindirect and immediate access is provided to the arteries in and aroundthe heart and other major organs and organ systems.

Another effective method of administering the composition is by directcontact with, for example, bone marrow through an incision or some otherartificial opening into the body. Compositions may also be administeredto the nasal passages as a spray. Arteries of the nasal area provide arapid and efficient access to the bloodstream and immediate access tothe pulmonary system. Access to the gastrointestinal tract, which canalso rapidly introduce substances to the blood stream, can be gainedusing oral, enema, suppository, or injectable forms of administration.Compositions may be administered as a bolus injection or spray asappropriate. Compositions may be given sequentially over time(episodically) such as every two, four, six or eight hours, every day(QD) or every other day (QOD), or over longer periods of time such asweeks to months. Compositions may also be administered in atimed-release fashion such as by using slow-release resins and othertimed or delayed release materials and devices. Orally activecompositions are more preferred as oral administration is usually thesafest, most convenient and economical mode of drug delivery. However,direct injection may be preferred when immediate access to the bloodsystem is desired.

Treatments to the patient may be therapeutic or prophylactic.Therapeutic treatment involves administration of one or morecompositions of the invention to a patient suffering from one or moresymptoms of the disorder. Symptoms typically associated with globindisorders include, for example, anemia, tissue hypoxia, organdysfunction, abnormal hematocrit values, ineffective erythropoiesis,abnormal reticulocyte count, abnormal iron load, splenomegaly,hepatomegaly, impaired peripheral blood flow, dyspnea, increasedhemolysis, jaundice, anemic crises and pain such as angina pectoris.Relief and even partial relief from one or more of these symptomscorrespond to an increased life span or simply an increased quality oflife. Further, treatments that alleviate a pathological symptom canallow for other treatments to be administered.

Prophylactic treatments involve pulsed administration of a compositionto a patient having a confirmed or suspected blood disorder withouthaving any overt symptoms. For example, otherwise healthy patients whohave been genetically screened and determined to be at high risk for thefuture development of a blood disorder may be administered compositionsof the invention prophylactically. Administration can begin at birth andcontinue, if necessary, for life. Both prophylactic and therapeutic usesare readily acceptable because these compounds are generally safe andnon-toxic.

It will be appreciated that the amount of the ecPNA-containingcompositions of the invention required for use in treatment will varywith the route of administration, the nature of the condition for whichtreatment is required, and the age, body weight and condition of thepatient, and will be ultimately at the discretion of the attendantphysician or veterinarian. These compositions will typically comprise atherapeutically effective amount of the compositions of the invention.Preliminary doses can be determined according to animal tests, and thescaling of dosages for human administration can be performed accordingto art-accepted practices.

Keeping the above description in mind, typical dosages of an ecPNAmolecule of the invention for ex vivo or in vitro use range from about 1μM to about 10 μM. Typical dosages of an ecPNA molecule of the inventionfor in vivo use range from about 0.1 mg/kg to about 100 mg/kg.

Keeping the above description in mind, typical dosages of cells (e.g.,bone marrow cells or peripheral blood cells) that have been treated withan ecPNA composition of the invention ex vivo for subsequentadministration to a patient range from about 1×10⁵ to about 1×10⁷ cellsper kg of body weight. In a preferred embodiment, the dosage is about1×10⁶ cells/kg body weight.

Combination Treatments

The present invention also encompasses combination treatments, whereinan additional agent or agents can be administered to a patientconjointly with an ecPNA of the invention (e.g., in the same compositionas the ecPNA or in separate compositions, at the same or differentsites, at the same or different times, and for the same or differentduration of time). In some embodiments, the additional agent or agentsare effective for enhancing the desired effect of the ecPNA on geneexpression.

Non-limiting examples of such combination treatments for β-globindisorders include conjoint administration of a γ-globin-specific ecPNAwith a second agent such as, for example, hydroxyurea, a short chainfatty acid (SCFA) inducer (e.g., butyrate), 5-azacytidine, or a histonedeacetylase inhibitor (e.g., suberoylanilide hydroxamic acid [SAHA]).For example, a pulsed butyrate regimen was shown to work best toeffectively increase γ-globin levels in patients who already exhibited aslightly higher baseline level (≧2%) of fetal hemoglobin (HbF)production. [See, Atweh, G. F. et al. Sustained induction of fetalhemoglobin by pulse butyrate therapy in sickle cell disease. Blood 93,1790-1797 (1999).]

In certain embodiments, ecPNA molecules are administered to source cellsin combination with one or more small molecules for the induction of iPScells. For example, small molecules that rearrange chromatin can beadministered to increase the efficiency of iPS cell generation. [See,Abujarour and Ding; (2009) Genome Biology 10: 220; Lin et al. (2009)Nature Methods 6, 805-808.]

In other embodiments, ecPNA molecules can be administered to cells withone or more cytokines or other active agents that increase theefficiency of iPS cell generation. Suitable cytokines or other activeagents are readily determined by one of ordinary skill in the art.

EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples constitute preferred modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Design, Synthesis, and Storage of PNA

A previous study that used PNA to alter γ-globin expression relied on atriplex-forming “clamp” design that forms a Hoogstein PNA/DNA/PNA triplehelix. [See Wang, G. et al., Peptide nucleic acid (PNA) binding-mediatedinduction of human gamma-globin gene expression. Nucleic Acids Res 27,2806-2813 (1999); and Pooga, M., Land, T., Bartfai, T. & Langel, U., PNAoligomers as tools for specific modulation of gene expression. BiomolEng 17, 183-192 (2001).] However, this design has significantlimitations, as it requires the use of base analogues that are not allcommercially available and the presence of homopurine/homopyrimidinesequences at the target site. [See Kaihatsu K., Janowski, B. A. & Corey,D. R. Recognition of chromosomal DNA by PNAs. Chem Biol 11, 749-758(2004).] Further, the possible number of DNA targets was significantlylimited using this approach.

In the present invention, it was desired to streamline this approach bydesigning short, complementary single stranded PNAs which function viastrand invasion and which can accommodate a wider range of DNA targets.By dividing the proximal γ-globin promoter (−202 to +33; SEQ ID NO: 41)into fifteen segments, four (4) 12- to 15-mer sequences suitable for PNAsynthesis were identified that correspond to the γ-globin promoter atpositions-150, -116, -78, -7, relative to the transcription start site(FIG. 1A). All PNAs were synthesized by Biosynthesis Inc. (Lewisville,Tex.) and have the following structures: PNA7:Biotin-OO-TGTGGAACTGCTGAA-O-k (Biotin-OO-(SEQ ID NO: 2)-O-k); PNA78:Biotin-OO-TACTCTAAGACTATT-O-k (Biotin-OO-(SEQ ID NO: 1)-O-k); PNA116:Biotin-OO-GGCTATTGGTCAAGGC-k (Biotin-OO-(SEQ ID NO: 3)-k); PNA150:Biotin-OO-GAGTTAGCCAGG-O-k (Biotin-OO-(SEQ ID NO: 4)-O-k); PNA78/NLS:Biotin-OO-TACTCTAAGACTATT-O-PKKKRKV (Biotin-OO-(SEQ ID NO: 1)-O-(SEQ IDNO: 12)); PNA78/TAT: Biotin-OO-TACTCTAAGACTATT-O-YGRKKRRQRRR(Biotin-OO-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)); MUT PNA78/TAT:Biotin-OO-TACTATAAAACTATT-O-YGRKKRRQRRR (Biotin-OO-(SEQ ID NO:88)-O-(SEQ ID NO: 6)); VP2/PNA78/TAT:Bio-OO-DFDLDMLGDFDLDMLG-O-TACTCTAAGACTATT-O-YGRKKRRQR (Bio-OO-(SEQ IDNO: 26)-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)); Bio-ATF/PNA78/TAT:Bio-OO-CGSDALDDFDLDML-O-TACTCTAAGACTATT-O-YGRKKRRQRRR (Bio-OO-(SEQ IDNO: 27)-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)); and ATF-Bio/PNA78/TAT:H₂N-CGSDALDDFDLDML-Biotin-O-TACTCTAAGACTATT-O-YGRKKRRQRRR ((SEQ ID NO:27)-Biotin-O-(SEQ ID NO: 1)-O-(SEQ ID NO: 6)). In the above structures,“O” represents the stable polyether linker, AEEA(2-aminoethoxy-2-ethoxyacetic acid). As used throughout the presentdisclosure, “Bio” is the abbreviated form of “biotin” are usedinterchangeably. All TAT amino acids were the D-isomer form. PNAs weredissolved in sterile distilled/deionized water and stored at 4° C., andheated at 50° C. for 5 minutes just before any application to preventaggregation.

Example 2 Magnetic Pull-Down Assay

A novel assay was developed to monitor specific binding of PNA moleculesto their cognate DNA site (FIG. 1B). 2 ng of ³²P-labelleddouble-stranded DNA oligonucleotide (designed at regions of theγ-promoter centered at each of the four PNA target sites) was incubatedwith various molar excess (0× to 1000×) of PNA at 37° C. overnight in afinal volume of 15 uL with 10 mM Tris and 1 mM of EDTA. The resultantmaterial was incubated with equal amount of Dynabeads M280-streptavidin(DYNAL biotech) for 15 seconds at 25° C. Beads had been prepared bywashing twice with BW buffer (10 mM Tris, pH7.5, 1 mM EDTA, 2.0M NaCl).The beads were pulled down with a Dynal MPC-S magnetic particleconcentrator and washed twice with BW buffer. Samples were finallysuspended in 50 uL BW buffer, and radioactivity was counted for 10minutes in a scintillation counter to yield the average counts perminute (cpm) taken for each sample, which was then divided by the cpm ofthe input (1 uL of the radio-labeled oligo).

It was found that the ability of each PNA to bind and discriminatebetween wild type and mutant oligos varied considerably. In the tests,PNA150 or PNA7 did not discriminate or bind well to their target sitesunder these conditions. However, PNA116 and PNA78 bound their target DNAoligo in proportion to their input. Even though PNA116 has the bestrecovery rate of its wild type DNA target, it also bound to the mutantDNA target to an unacceptable extent. On the other hand, PNA78 exhibitedthe best discrimination (˜25-fold) and specificity in recognizing itstarget sequence.

Example 3 KMnO₄ Probing and Footprinting

To further confirm the observation in the magnetic-pull down assay,permanganate (KMnO₄) probing of the PNA78/DNA interaction in solutionwas carried out (FIG. 1C). In a modification of a published protocol[Bentin, T., Hansen, G. I. & Nielsen, P. E., Measurement of PNA bindingto double-stranded DNA. Methods Mol Biol 208, 91-109 (2002)], 1 ng of asingly ³²P-end-labeled, gel purified RsaI/XhoI fragment derived from thep250 plasmid that contains the γ-globin promoter region −299 to +37 wasincubated in a volume of 15 μL with a final concentration of 10 mM Trisand 1 mM EDTA for 30 minutes at 37° C. PNA was added at a finalconcentration of 50 uM for further incubation overnight at 37° C. 1 uL20 mM KMnO₄ was added to the reaction, and after 15 s 10 uL stop buffer(1.5 M sodium acetate, 1 M 2-mercaptoethanol, Ph 7.0) was added. 2.5×vol of 96% ethanol was added and the DNA collected by centrifugation at13000 rpm for 15 minutes. The supernatant was removed and the DNA pelletdissolved in 100 uL 10% piperidine followed by incubation at 90° C. for20 minutes. The lympholized DNA sample was resuspended in 8 μL FA buffer(80% deionized fomamide, 10 mM EDTA, 0.25% xylenecyanol FF, 0.25%bromphenol blue). DNA was denatured at 90° C. for 2 minutes, chilled onice, and then electrophoresed on thin 10% polyacrylamide gels containing7 M urea. The dried gel was then exposed for autoradiography. KMnO₄probing gives single-base pair resolution and makes it easy to map thePNA78 target site on the γ-promoter when co-electrophoresed adjacent toa separate reaction that generates a partial sequence ladder.

The results demonstrated that PNA78 binds specifically to its targetregion within the γ-globin promoter fragment, with residues at the 5′end of its interaction most accessible to chemical cleavage. Based onthe results of the in vitro studies, PNA78 was used for the remainder ofthe experiments described in the present Examples.

Example 4 Cellular and Nuclear Localization of PNA

In order to alter transcription, it is necessary for the PNA to reachand enter the cell nucleus. The basis for cellular uptake of PNA is notyet fully understood, although it may be related to passive diffusion[see e.g., Pooga, M., Land, T., Bartfai, T. & Langel, U. PNA oligomersas tools for specific modulation of gene expression. Biomol Eng 17,183-192 (2001)], particularly as unmodified and slightly modified PNAshave been successfully delivered directly to cells without the use oftransfection reagents and protocols. [See Nielsen, P. E., Peptidenucleic acid: a versatile tool in genetic diagnostics and molecularbiology. Curr Opin Biotechnol 12, 16-20 (2001); and Sei, S. et al.,Identification of a key target sequence to block human immunodeficiencyvirus type I replication within the gag-pol transframe domain. J Virol74, 4621-4633 (2000).] In order to ascertain whether the PNA moleculesefficiently enter an erythroid cell, varying amounts of PNA78 wereincubated for different lengths of time with erythroleukemic K562 cells(ATCC deposit number CCL243) and entry and localization was monitored byfluorescent microscopy after incubation with streptavidin-FITC. K562cells are human erythroid leukemia cells that display similarcharacteristics of fetal erythrocytes, because they express high levelsof γ-globin and no or very low levels of beta globin. [SeeStamatoyannopoulos, J. A. & Nienhuis, A. W., Therapeutic approaches tohemoglobin switching in treatment of hemoglobinopathies. Annu Rev Med43, 497-521 (1992).]

PNA efficiently entered cells after a simple overnight incubation ingrowth media with an optimal concentration of 10 uM (FIG. 2A). Based onpublished studies, concentrations of PNA as high as 30 uM are not toxicto cultured cells. [See Cutrona, G. et al., Effects in live cells of ac-myc anti-gene PNA linked to a nuclear localization signal. NatBiotechnol 18, 300-303 (2000).] Although the efficiency of cell entrywas high (over 90%) after an overnight incubation, in all cases noevidence of nuclear entry was observed, as all of the PNA moleculesremained visible as punctate spots in the cytoplasm.

Cell Culture

COS7 cells were maintained in DMEM (Cellgro®, Mediatech Inc. (Manassas,Va.)) and K562 cells were maintained in RPMI 1640 medium (Gibco®,Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum,penicillin and streptomycin. CID-dependent wild type β-YAC mouse bonemarrow cells (MBCs) [see Blau, C. A. et al., γ-Globin gene expression inchemical inducer of dimerization (CID)-dependent multipotential cellsestablished from human β-globin locus yeast artificial chromosome(β-YAC) transgenic mice. J Biol Chem 280, 36642-36647 (2005)] werecultured in the presence of AP20187 dimerizer (100 nM; AriadPharmaceuticals, Cambridge, Mass.) in Isocove's modified Dulbecco'smedium containing 10% fetal bovine serum, penicillin, and streptomycin.

Delivery of PNA to Cells

PNA was introduced into actively growing COS7 cells. For suspensioncells, 3×10⁵ K562 cells or β-YAC MBCs were resuspended with 200 uLOPTI-MEM, and plated on poly-lysine coated 8-well chambered cover glassslides (Nunc) and allowed to attach to the slide overnight. In allcases, PNA was added to a final concentration of 5 uM or 10 uM alongwith Strepavidin-FITC to a final concentration of 5 ug/mL. Based onpublished studies, concentrations of PNA as high as 30 uM are not toxicto cultured cells. [See Cutrona, G. et al., Effects in live cells of ac-myc anti-gene PNA linked to a nuclear localization signal. NatBiotechnol 18, 300-303 (2000).] After 4 hours, 300 uL RPMI 1640 and FBSwas added to a final concentration of 10%.

Confocal Analysis and Live Cell Imaging of Cellular Localization of PNAs

A live cell imaging protocol was developed to monitor cellularlocalization of chimeric PNA molecules. At 16 hours post PNA-treatment,DRAQ5 (Biostatus Limited, UK, [see Martin, R. M., Leonhardt, H. &Cardoso, M. C., DNA labeling in living cells. Cytometry A 67, 45-52(2005)]) was added to cells to a final concentration of 1 μM, andincubated at room temperature for 30 minutes; media was discarded andcells were gently washed twice with 1×PBS. 100 uL 1×PBS was then addedto each chamber for further observation for confocal microscopy. DIC,DRAQ5 (staining cell nucleus), and FITC images were collected on theZEISS LSM-510 META Confocal Microscope.

To help more easily detect subcellular localization of the PNA78/TAT orPNA78/NLS, initial tests were performed with COS7 cells (FIG. 2B). Theresults showed that, after only a 2-hour incubation, the PNA/TATmolecules not only entered the cells but also proceeded to the nucleus.

This procedure was then performed with the K562 erythroid cell line.However, because these normally grow in suspension, they were firstattached to poly-L-lysine coated plates to enable easier visualizationof the signal localization. As before, the PNA molecules were directlyadded to K562 cells in serum-free media along with streptavidin-FITC,and their localization was monitored by confocal microscopy withoutfixation (FIG. 2C). Easily visible bright positive signals were presentin the cell nucleus both in PNA78/TAT and PNA78/NLS treated cells;however, the distribution and efficiency was significantly higher inPNA78/TAT treated cells. These results were highly encouraging, asefficiencies of cell entry were always greater than 50% and sometimesapproached 95%. Based on these experiments, it was concluded that PNAmolecules attached to a TAT sequence at its 3′ end provide a consistentand efficient means of directing the PNA cargo to the nucleus.

Example 5 In Vivo Target Binding of PNA78 in K562 Cells Using a NovelChromatin Association Assay

To determine whether PNA78 can interact with its target site in vivo, amodification of the chromatin immunoprecipitation assay was developed.This assay took advantage of the biotin label already incorporated intothe 5′ end of the PNA molecule and was based on the standard ChIP assaywhereby streptavidin-magnetic beads (rather than antibodies) are used topull-down the PNA78/DNA complex after the normal series of PNA/cellincubation, formaldehyde cross-linking, and chromosomal DNA shearingsteps (FIG. 3A). Along with the “no addition” negative control, avariant of PNA78 that contain two point mutations (MUT PNA78) wassynthesized and used it as an additional negative control along with a“TAT alone” control.

As the PNA/DNA interaction is not protein-based, a novel chromatinassociation assay that does not utilize antibodies, but rather relies onthe biotin moiety in the PNA molecule as a tag for its isolation withstreptavidin beads was developed. This assay incorporates elements ofchromatin [see Siatecka, M., Xue, L. & Bieker, J. J., Sumoylation ofEKLF promotes transcriptional repression and is involved in inhibitionof megakaryopoiesis. Mol Cell Biol 27, 8547-8560 (2007)] andbiotinylated protein [see Viens, A., Mechold, U., Lehrmann, H.,Harel-Bellan, A. & Ogryzko, V., Use of protein biotinylation in vivo forchromatin immunoprecipitation. Anal Biochem 325, 68-76 (2004)]immunoprecipitation protocols. PNA 78/TAT or mutant (MUT) PNA 78/TATwere added to 10⁷ K562 cells to a final concentration of 10 μM. After 36hours, a portion of the cells were analyzed and quantified for PNAincorporation efficiency by confocal analysis of live cells (as above),and the rest were harvested and cross-linked with 0.4% formaldehyde(Fisher Scientific, F79-500) for 10 minutes in room temperature thenquenched by addition of glycine in the final concentration of 0.125 Mfor another 10 minutes. Cells were then washed twice in cold PBS,pelleted at 3000 rpm and suspended in 500 μL SDS buffer (50 mM Tris atpH 8.1, 05% SDS, 100 mM NaCl, 5 mM EDTA and protease inhibitors) andincubated 10 minutes on ice. Cells were then pelleted by centrifugationand resuspended in 1.6 ml IP buffer (0.3% SDS, 1.1% Triton X-100, 1.2 mMEDTA, 16.7 mM Tris pH8.1, 167 mM NaCl and protease inhibitor) anddisrupted by sonication (power 4, 21% duty, 30 seconds each time for 10times with 2 minute-pulse on ice) yielding genomic DNA fragments of˜1000 bp. Lysates were collected at 13000 rpm for 30 minutes and diluted1:5 in IP buffer and incubated with 40 uL streptavidin MagneSphereParamagnetic particles (Promega Cat#Z5481) for 2 hours. MagneticParticle Concentrator (Dynal MPCS) was used to collect the MagneSphereand its bound material, which was washed five times with LiCl buffer(100 mM Tris pH8.0, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid). Beadswere finally resuspended in 150 μL 300 mM NaCl at 65° C. overnightfollowed by incubation at 42° C. for 2 hours with 20 μL protein kinase K(20 mg/mL). DNA was purified by phenol/chloroform extraction andresuspended in 20 μL ddH2O.

After 16 hours of incubation of K562 cells, any formed PNA-chromatincomplex was cross-linked and cells were disrupted and chromatin wassheared by sonication. Since all the PNAs were linked to biotin, thecomplex formed between PNA and the bound chromatin was pulled down andisolated by binding to streptavidin-conjugated magnetic beads. The DNAwas purified for sequence identification by quantitative PCR. To moreprecisely quantitate the extent of PNA entry into the nucleus, a portionof the cells from each treatment was collected, stained with the DRAQ5live-cell nuclear stain, attached to chamber-slides, and inspected underconfocal microscopy to compare PNA and TAT nuclear entry efficiency. Theresults (FIG. 3B) show that while the two PNAs and TAT were taken up bycells with a similar efficiency, only wild-type (WT) PNA78 interactedwith its target sequence within the γ-globin promoter to a level greaterthan the non-specific MUT PNA78 and TAT alone, which had values that arenot higher than the ‘no addition’ control. This data confirmed that WTPNA78 has target binding specificity with the endogenous γ-globinpromoter in vivo.

Example 6 PNA Conjugated with a Minimum Activation Domain (AD) AltersTranscriptional Activity in K562 Cells

The above experiments established that WT PNA78 is able to enter thecell nucleus and bind to its target sequence at the γ-globin promotersite in vivo. To assess whether WT PNA78 can alter transcription, atransient assay was used. A γ-globin luciferase reporter was transectedinto K562 cells followed by exposure of the cells to TAT, PNA78/NLS, orPNA78/TAT. Although TAT alone had no effect on the basal activity of thereporter, PNA78/NLS exerted a mild, and PNA78/TAT a stronger,superactivation effect on its expression (FIG. 4A). As predicted,mutation of the PNA78 target site on the γ-promoter negated its abilityto be superactivated by PNA78/TAT. However, the basal activity of theγ-promoter was dramatically impaired (FIG. 4B), suggesting that the DNAsequence complementary to PNA78 encodes an important γ-promoter element.

It was then examined whether the potential of WT PNA78 to altertranscriptional activity in vivo can be enhanced by linking it to atranscriptional activator motif. The VP2 minimal activation domain (AD)was chosen for the first set of experiments. The VP2 minimal activationdomain is a highly acidic 16 amino acid sequence (MLGDFDLDMLGDFDLD; SEQID NO: 30) derived from the herpes simplex virus C-terminustransactivation domain of VP16. [See Ansari, A. Z., Mapp, A. K., Nguyen,D. H., Dervan, P. B. & Ptashne, M., Towards a minimal motif forartificial transcriptional activators. Chem Biol 8, 583-592 (2001).] VP2is a very potent artificial transactivator in vitro when linked to aDNA-binding protein domain. [See Arora, P. S., Ansari, A. Z., Best, T.P., Ptashne, M. & Dervan, P. B., Design of artificial transcriptionalactivators with rigid poly-L-proline linkers. J Am Chem Soc 124,13067-13071 (2002).] This amino acid sequence was linked to PNA78/TAT atits 5′ domain to yield VP2/PNA78/TAT. This molecule retains the biotinlabel at the beginning of the VP2 sequence for monitoring purposes. Theability to superactivate the γ-globin/luciferase reporter in transfectedK562 cells was tested, but there was little effect beyond that seen withPNA78/TAT. Then an artificial luciferase reporter plasmid wasconstructed with four tandem repeats of PNA78 target sequence upstreamof the minimal SV40 promoter and its activity in erythroid K562 cellline and in the non-erythroid 293T cell line (ATCC deposit numberCRL11268) was tested. Three hours post-transfection, PNA78TAT orVP2/PNA78/TAT were added to cells in serum-free media. A portion of thecells were also inspected under confocal microscopy afterstreptavidin-FITC treatment to normalize PNA uptake efficiency. It wasfound that the luciferase activity increased by 2.3-fold in cellstreated with VP2/PNA78/TAT compared to non-treated cells, a strongereffect than seen with PNA78/TAT and not observed at all in 293T cells(FIG. 4C). This demonstrated that the attachment of a transactivationdomain to the PNA78/TAT molecule enhances its ability to activatetranscription in the K562 fetal-like erythroid environment

Plasmids

The γ-globin reporter contained a 1.5 Kb Human HS2 fragment upstream ofthe −299 to +37 human γ-globin promoter in Promega's (Madison, Wis.)pGL2 Basic plasmid. [See Caterina, J. J., Donze, D., Sun, C. W.,Ciavatta, D. J. & Townes, T. M., Cloning and functional characterizationof LCR-F1: a bZIP transcription factor that activateserythroid-specific, human globin gene expression. Nucleic Acids Res 22,2383-2391 (1994).] A modified pGL2-promoter vector from Promega with oneor four copies of the PNA78 target sequence(CCGGTTGACCAATAGTCTTAGAGTATCC; SEQ ID NO: 40) upstream of the minimalSV40 promoter was also generated by synthesis of the oligonucleotidefollowed by ligation into the XmaI and XhoI sites of the vector.

Transfection

Transfection of K562 cells was performed using the DMRIE lipofectionmethod (Gibco, Invitrogen, Carlsbad, Calif.). K562 cells were countedand 1×10⁵ cells were resuspended in 0.1 mL OPTI-MEM in a 12-well plate(BD Falcon, Franklin Lakes, N.J.). Experiments were all done intriplicates. 1.5 ng Renilla plasmid (Promega Corporation, Madison, Wis.)and 0.5 g reporter construct were added to each well. After 2 hours oftransfection, PNAs were added to a final concentration of 10 μM. 2 hourslater, 1 ml RPMI and a final concentration of 10% FBS were added to eachwell. This was followed by incubation at 37° C. for 36 hours.

Luciferase Reporter Assay

36 hours after PNA addition, K562 cells were collected by centrifugationat 2500 rpm. Media was discarded and cells were washed once with 1×PBSand resuspended in 250 μL 1× passive lysis buffer (PLB) (Promega). Tubesthat contained cells were put on a rocker platform for 10 minutes atroom temperature. Cells were then centrifuged at 13000 rpm andresuspended in 200 μL 1×PLB, loaded on a 96 well plate and furtheranalyzed with a Turner Biosystem Microplate Luminometer. [See Siatecka,M., Xue, L. & Bieker, J. J., Sumoylation of EKLF promotestranscriptional repression and is involved in inhibition ofmegakaryopoiesis. Mol Cell Biol 27, 8547-8560 (2007).] Luciferase valueswere divided by the Renilla value from the same sample after it had beendivided by 1000. To minimize the differences in between loading, eachsample was loaded in three different wells and the average of three wascalculated.

Example 7 PNA-AD Changes the Globin Expression Pattern in Mouse BoneMarrow Cells

The above tests had been directed at a non-chromatinized DNA targetpromoter that is already highly active prior to any treatment. It wasthus important to test whether the PNA constructs can activate a dormantγ-globin promoter in an erythroid cell that has already switched off itsexpression in favor of adult If-globin. To test this, a line of mousebone marrow cells engineered to carry a yeast artificial chromosome thatcontains the complete human β-like globin locus (β-YAC) was used. [SeeBlau, C. A. et al. γ-Globin gene expression in chemical inducer ofdimerization (CID)-dependent multipotential cells established from humanβ-globin locus yeast artificial chromosome (β-YAC) transgenic mice. JBiol Chem 280, 36642-36647 (2005).] These adult bone marrow cells haveshut off human fetal γ-globin expression and solely express human adultβ-globin (in addition to the endogenous mouse adult β-globin). The largeβ-YAC is properly controlled during murine development in that humanγ-globin is expressed in the yolk sac but switches expression to humanβ-globin at the same time as the endogenous mouse β-globin. [SeePeterson, K. R. et al., Use of yeast artificial chromosomes (YACs) forstudying control of gene expression: correct regulation of the genes ofa human beta-globin locus YAC following transfer to mouseerythroleukemia cell lines. Proc Natl Acad Sci USA 90, 11207-11211(1993).] The fact that these cells are responsive to a known γ-globininducer (5-azacytidine) makes them particularly clinically relevant.[See Blau, C. A. et al., supra.]

To investigate whether PNA78 attached to an activation domain canreactivate the endogenous γ-globin gene in these mouse bone marrowcells, its expression was compared in cells treated with two differentminimal AD attached to WT PNA78: VP2, and another minimal activationdomain, ATF14 (CGSDALDDFDLDML; SEQ ID NO: 27), which is also highlyacidic and has been shown to be a promising candidate as an artificialtranscription factor in vitro and in vivo. [See Qiu, C., Olivier, E. N.,Velho, M. & Bouhassira, E. E., Globin switches in yolk sac-likeprimitive and fetal-like definitive red blood cells produced from humanembryonic stem cells. Blood 111, 2400-2408 (2008).] In addition, toaddress whether the relative position of the AD and biotin may affectits transactivational ability or even the ability to visually monitorits presence, two PNA chimeric variants with ATF14 were generated: one(analogous to PNA/VP2 construct) with the biotin at the 5′ end (aminoterminus) of the molecule (Bio-ATF/PNA78/TAT), and one with the biotininserted between ATF and PNA78 (ATF-Bio/PNA78/TAT).

After treatment, cells were harvested and mRNA was extracted for reversetranscriptase (RT)-quantitative polymerase chain reaction (qPCR) usingtwo different sets of published primers that recognize sequences withinthe γ-globin gene. [See Blau, C. A. et al., supra and Qiu, C., et al.,supra]. The results (FIG. 5A and FIG. 5B) show that althoughVP2/PNA78/TAT increased γ-globin levels about 2-fold, ATF-Bio/PNA78/TATis the most effective at increasing γ-globin expression, attaining a7-fold increase in comparison to cells treated with PNA78/TAT alone. Itis of interest that placement of the biotin is critical, as placing itat the extreme 5′end (Bio-ATF/PNA78/TAT) precluded its ability toactivate γ-globin expression. It was also found that γ-globin protein,which is not detectable in the untreated cells, is expressed inATF-Bio/PNA78/TAT cells in the cytoplasm at levels approaching that seenin uninduced K562 cells (FIG. 5C). From these studies it was concludedthat it is possible to design a chimeric molecule containing peptidenucleic acids linked to cell entry/localization and activation aminoacid sequences that can bind and directly activate transcription of aspecific endogenous target gene in the cell.

The ability of the chimeric PNA molecule's ability to stimulate γ-globinexpression was further tested in human mobilized peripheral blood CD34+cells. When incubated in a two-step serum-free culture system, theseprogenitors differentiated to phenotypically and morphologically matureerythroid cells (FIG. 1 IA, FIG. 11B). Changes in cell surfaceexpression were already apparent between days 2-4 of culture (FIG. 12A),and the onset of adult 6-globin RNA and protein expression begins by day2 (FIG. 12B, FIG. 12C). As a result, peripheral blood CD34+ cellscultured for two days were used for further testing.

A mismatched PNA78 variant (ATF-Bio/MUT-PNA78/TAT) was used that wasdesigned based on MUT-PNA as an additional negative control. PurifiedCD34+ cells were cultured, treated at the end of day 2 with the variousPNA chimeras or with no PNA, and harvested 16 hours later for analysis.The results show that only cells treated with ATF-Bio/WT-PNA78/TATincrease γ-globin expression (by almost 5-fold compared to untreatedcells) (FIG. 13A), and that this leads to γ-globin protein accumulationsolely in those cells (FIG. 13B). Visual analysis of ≧100 cells perfield from the same experiment indicates that 22% of cells treated withATF-Bio/WT-PNA78/TAT were positive; all other samples were 0% positive(FIG. 13B). Use of a novel flow cytometric analysis ofstreptavidin-FITC-positive (i.e., PNA-positive) cells not onlydemonstrated a high level of PNA incorporation into the cultured CD34+cells, but also confirmed that cell death caused by PNA (measured bydual monitoring of FITC+/7-AAD+) was always below 0.5% (FIG. 13C). InFIG. 13C, the percent dead cells (7-AAD-positive) among all PNA-positivecells (>98% FITC+ as boxed in the PNA78/TAT example on the left) isindicated above the 7-AAD gate and was no higher than untreated cells.In FIG. 13C, FITC+/7-AAD+ (i.e., percent dead cell) results are alsoshown for CD34+ cells treated with ATF-Bio/MUT-PNA78/TAT orATF-Bio/WT-PNA78/TAT. Expression profiles of phenotypic markers (CD34,CD36, CD235a, and CD71) were unaltered as well (FIGS. 14A-14C),demonstrating that there was no erythroid differentiation abnormalitycaused by PNA treatment. As shown in FIG. 14A-14C, all cells has similarforward scatter (“FSC”) and side scatter (“SSC”) profiles (FIG. 14A) andexpressed the same level of CD34, CD36, and CD235a markers (FIG. 14B)even after exposure to the different PNA78 variants. In a separateexperiment, H-PB cells from day 4 of culture were analyzed by flowcytometry 24 hours after treatment and show the same level of CD235a andCD71 expression irrespective of variant PNA78 exposure (FIG. 14C).

Quantitative PCR Analysis

One microliter of DNA was used in 20 μl of quantitative PCR reactionusing a Quantitect SYBR Green PCR kit from Qiagen (Valenica, CA) and thepresence of γ-globin promoter sequences was quantified with an ABI PRISM7900HT and SDS software. [See Lohmann, F. & Bieker, J. J. Activation ofEklf expression during hematopoiesis by Gata2 and Smad5 prior toerythroid commitment. Development 135, 2071-2082 (2008).] Each reactionwas performed in triplicate. Primers used for the analysis were:Gamma-promo-fwd: AGCCTGACAAGGCAAACTIGA (SEQ ID NO: 31); Gamma-promo-rev:CCCTGGCCTCACTGGATACTCT (SEQ ID NO: 32).

Quantitative Globin Expression Analysis

Total RNA from β-YAC MBCs were isolated with a RNeasy micro kit (Qiagen)following the manufacturer's instructions. RNA concentrations werequantified using a Nanodrop. Reverse transcription of RNA was performedwith Promega's Reverse Transcription System and expression levels werequantified using a Quantitect SYBR Green PCR kit from Qiagen inconjunction with an ABI PRISM 7900HT and SDS software [see Lohmann, F. &Bieker, J. J. supra]Q-PCR results for globin expression were normalizedby 2−[(Ct γ−Ct β)−(Ct γ [no PNA]−Ct β[noPNA])][see Livak, K. J. &Schmittgen, T. D., Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods25, 402-408 (2001)] divided by the PNA incorporation efficiency(determined from the live cell image data). Each reaction was performedin triplicate.

The PCR primer sequences were derived from two studies [see Blau, C. A.et al. and Qiu, C., Olivier, E. N., Velho, M. & Bouhassira, E. E. Globinswitches in yolk sac-like primitive and fetal-like definitive red bloodcells produced from human embryonic stem cells. Blood 111, 2400-2408(2008)]: for globin expression in MBCs: (primer set 1) humangamma-globin forward: ACCGTTTGGCAATCCATTrC (SEQ ID NO: 33), reverse:TATTGCTTGCAGAATAAGCC (SEQ ID NO: 34); (primer set 2) human gamma-globinforward: GACCGTITGGCAATCCATTTC (SEQ ID NO: 35), reverse:TATTGCTTGCAGAATAAGCC (SEQ ID NO: 36). Both studies used the same set ofhuman β-globin primers: forward: ACACAACTGTGTTCACTAGCAACCTCA (SEQ ID NO:37), reverse: GGTTGCCCATAACAGCATCAGGAGT (SEQ ID NO: 38).

Immunofluorescent Detection of γ-Globin Protein

At 48 hours post-PNA treatment, 100K MBC-βYAC cells were collected andspun onto frosted slides (Fisher) in a Shandon Cytospin 2 centrifuge at500 rpm for 5 minutes. All steps were carried out at room temperatureexcept when indicated. Cells were fixed for 4 minutes in ice-coldacetone/methanol (1:1) on ice and rinsed twice with 2% BSA/PBS. Sampleswere then incubated with blocking solution (10% FCS, 0.05% NP40/PBS) for30 minutes followed by a two-hour incubation with hemoglobin γ antibody(Santa Cruz Biotechnology, sc-21756) diluted 1:100 in blocking solution.Slides were washed 3× with 2% BSA, 0.05% NP40/PBS and then incubatedwith goat anti-mouse secondary antibody conjugated to Alexa 568(Invitrogen) at a 1:50 dilution for one hour in a dark chamber. Slideswere then washed 3× with 2% BSA/PBS with a final rinse in PBS andmounted with DAPI-containing Vectashield. DAPI and Alexa 568 images werevisualized on a Zeiss spinning disc confocal microscope (PerkinElmer).

PNA Persistence Test

30000 K562 cells were suspended in OPTI-MEM and attached to the bottomof a 8-well L-polylysine coated cover glass chamber slide (Nunc) andtreated with ATF-Bio/PNA78/TAT in a final concentration of 10 uM andincubation at 37° C. 2 hours after PNA addition, Streptavidin-FITC wasadded to cells with a final concentration of 5 ug/mL followed by another2 hours of incubation at 37° C. Cells were inspected every day after 48hours post-PNA treatment under ZEISS LSM-510 META confocal microscopy.DRAQ5 was used to stain cell nucleus 30 minutes prior to each confocalobservation. Images of DRAQ5 and FITC were collected and compared withdata collected during previous days.

Human Erythroid Cell Culture

Erythroid cells were derived from adult human mobilized peripheral blood(H-PB) CD34⁺ cells (All Cells, LLC. Emeryville, Calif., USA) in atwo-step liquid culture system. In the first step, CD34⁺ cells wereplated at a density of 0.5×10⁶ cells/ml in expansion media consisting ofIscove's Modified Dulbeco Media (IMDM) supplemented with 1%penicillin/streptomycin, 1% L-Glutamine (Invitrogen, Grand Island, N.Y.,USA), 20 mM B-merchaptoethanol, 1% bovine serum albumin (BSA) Fraction V(Sigma, St. Louis, Mo., USA), 30% serum substitute BIT 9500 (Stem CellTechnologies, Vancouver, BC, Canada), 100 ng/ml Stem Cell Factor (SCF)(R&D Systems, Minneapolis, Minn., USA) and 4 U/ml Erythropoietin (EPO)(Amgen, Thousand Oaks, Calif., USA) and incubated at 37° C. in ahumidified 5% CO₂ incubator for seven days. In the second step, thecells were collected by centrifugation and replated in differentiationIMDM media supplemented with 1% penicillin/streptomycin, 1% L-Glutamine,20 mM β-mercaptoethanol, 1% BSA, 30% BIT 9500 and 4 U/ml EPO and allowedto differentiate for seven additional days.

Phenotypic Characterization of Human Erythroid Cells

Expression of cell surface antigens CD34, CD235a (glycophorin A) andCD36 was evaluated by flow cytometric analysis of immunolabelled cells.Fluorescein (FITC)-conjugated CD34, phycoerythrin (PE)-conjugated CD36and allophycocyanin (APC)-conjugated CD235a antibodies and theappropriate isotypes control were obtained from Becton Dickinson. Dataacquisition and analysis was performed using a BD FACSCanto II flowcytometer and FACS Diva software, respectively (Becton Dickinson,Mountain Blue, Calif., USA). Non-viable cells were excluded from flowcytometric analysis by gating based on positive staining with7-amino-actinomycin (7-AAD) (Sigma, St. Louis, Mo., USA). Erythroiddifferentiation was also monitored by Wright-Giemsa staining andmicroscopic analysis of cytospins.

Example 8 Use of Animal Model for Testing ecPNAs for Treatment of aβ-Globin Disorder

Efficacy of the γ-globin specific ecPNAs of the invention can be testedin a humanized mouse model of a β-globin disorder. The mice used in thismodel have been described. One suitable example is a humanized mousemodel of Cooley's Anemia (CA) which has been generated by targeted genereplacement in embryonic stem (ES) cells. In these mice, a delayedswitching human γ to β0 globin gene cassette (γβ0) is inserted directlyinto the murine β globin locus replacing both adult mouse β globingenes. The inserted human β0 globin allele has a mutation in the splicedonor site that produces the same aberrant transcripts in mice asdescribed in human cells. No functional human β globin polypeptidechains are produced. Heterozygous γβ0 mice suffer from microcyticanemia. Homozygous γβ0 mice switch from mouse embryonic globin chains tohuman fetal γ-globin during fetal life. When bred with human α globinknockin mice, homozygous CA mice survive solely upon human fetalhemoglobin at birth. [See, Yongliang Huo et al. et al. (2009) J. Biol.Chem. 284: 4889-4896.]

Bone marrow cells can be isolated from these humanized mice (expressingthe human globin genes) and cultured as described above. The γ-globinspecific ecPNAs of the invention can be transfected into these culturedbone marrow cells. The transfected cells (1 to 10×10) can be thenadministered to the transgenic mice via tail vein injection in 200 mlPBS and the efficacy of the transfected cells for treating the β-globindisorder can be assessed by determining the red blood cell count.

Example 9 Induction of iPS Cells from Human CD34+Peripheral Blood Cells

Mobilized peripheral blood cells are obtained as follows. A donorsubject is injected with 5 μg/kg/day for 3 days with Neupogen (G-CSF)manufactured by Amgen. On the fourth day, apheresis of 7 blood volumesis performed on a Cobe Apheresis machine, and 300 ml of blood iscollected. Using this method, the yield of CD34+ cells is about 1%. Forculture, mobilized peripheral blood CD34+ cells (Allcells, mPB014F) aremaintained in IMDM (Invitrogen) containing 15% fetal bovine serum(StemCell Technologies) supplemented with hSCF (100 ng/ml), hFlt3L (100ng/ml), and Interleukin-3 (20 ng/ml) (Peprotech).

CD34+ cells grown in culture for 4 days are then used for treatment withecPNA molecules. The cells are treated with a 10 μM cocktail of equalmolar concentrations of two ecPNA molecules. The first ePNA molecule hasthe structure: H₂N-CGSDALDDFDLDML-O-PNA₁-O-YGRKKRRQRRR, wherein PNA₁ hasa nucleic acid sequence that is complementary to a 12 bp sequence in the200 bp region upstream of the transcription start site of OCT4 gene (seeFIG. 7). The second ecPNA molecule has the structure:H₂N-CGSDALDDFDLDML-O-PNA₂-O-YGRKKRRQRRR, wherein PNA₂ has a nucleic acidsequence that is complementary to a 12 bp sequence in the 200 bp regionupstream of the transcription start site of SOX2 gene (see FIG. 8).

Once iPS cells begin to form in the cultures, embryoid bodies are formedby mechanically scraping confluent undifferentiated CD34 iPS cells intostrips and transferring the cells to 6-well, low attachment plates indifferentiation medium consisting of knockout DMEM (Invitrogen)supplemented with 20% fetal bovine serum (StemCell Technologies), 0.1 mMnon-essential amino acids (Invitrogen), 1 mM L-glutamine (Invitrogen),50 μg/ml ascorbic acid (Sigma), and 2 mg/ml human holo-transferrin(Sigma) and 0.1 mM β-mercaptoethanol (Sigma).

iPS cells are characterized by immunofluorescent microscopy followingimmunolabeling with TRA-1-60 (MAB4360, 1:200), TRA-1-81 (MAB4381,1:200), and SOX2 (AB5603, 1:500) all Chemicon, SSEA-4 (MC-813-70, 1:2),SSEA-3 (MC-631, 1:2) all Iowa, Tuj1 (1:500; Covance), α-fetoprotein(1:400; Dako), α-actinin (1:100; Sigma), OCT4 (C-10, SantaCruz, sc-5279,1:100), and NANOG (Everest Biotech EB06860, 1:100).

Example 10 Use of ecPNAs to Establish Induced Pluripotent Stem (iPS)Cells

The proximal promoter regions of the OCT4 and SOX2 genes (ie, a 200 bpDNA located upstream of their transcription initiation sites) weredivided into 15 base pair sequences against which complementary 15-merPNA sequences were designed. Four regions in each promoter were selectedfor synthesis (ie, eight PNA molecules in all; note that all PNAmolecules contained biotin for detection).

These PNAs west tested, along with a mutant variant of each (sixteentotal), for selective and specific association with their cognate DNAtarget by an in vitro binding assay. The results (FIG. 19, threeexperiments) show that PNAs Sox2-66 and Sox2-81 exhibited better bindingto wild-type relative to mutant SOX2 promoter sequences. Using a similarassay (FIG. 20, two experiments), PNAs Oct4-54 and Oct4-111 exhibitedreasonable discrimination of wild-type vs mutant OCT4 promoter sequences(note that binding was relatively higher than seen with SOX2, andOct4-100 had too high a background level for further consideration).

The best two PNA sequences for each of the two promoters formed thebasis for the design of ecPNAs for increasing the gene expression ofOCT4 and SOX2. The ecPNAs comprised TAT cell/nuclear entry and thetranscriptional activation (ATF) motifs, and a the OCT4- orSOX2-specific PNA sequence (four total), and are diagrammed in FIG. 21.

Each of the four ecPNA candidates are tested for efficient cell andnuclear entry and effective activation of OCT4 and SOX2 promoters incultured fibroblast (293T) cells. The four molecules efficiently enterthe 293T cell nucleus, and efficiently activate their specificdownstream target (OCT4 or SOX2).

Example 11 Evaluation of ecPNA Molecules that Repress Target GeneExpression

The TAT-PNA78-FOG12 ecPNA repressor was synthesized using the 12 aminoacid repression sequence from FOG12: MSRRKQSKPRQI (SEQ ID NO: 47).

This ecPNA was tested for cell and nuclear entry into live cells afterincubation with K562 cells. K162 cells are a human leukemic cell line,and was also used hereinabove for the evaluation of TAT-PNA78-ATF. Thesecells constitutively express γ-globin, and are particularly rueful fortesting repression of that gene by the TAT-PNA78-FOG12 ecPNA. FIG. 22shows that the TAT-PNA78-FOG12 ecPNA molecule entered the cells andtheir nuclei efficiently.

The TAT-PNA78-FOG12 ecPNA represses γ globin RNA and protein synthesis.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

While the compositions and methods of this invention have been describedin terms of specific embodiments, it will be apparent to those of skillin the art that variations may be applied to the compositions andmethods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the scope of theinvention a defined by the appended claims.

It is further to be understood that all values are approximate, and meprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. An embedded chimeric peptide nucleic acid (ecPNA) having the generalstructure:H₂N—X—B—Y—COOH wherein X is A or C and Y is A or C with the proviso thatwhen X is A, Y is C, and when X is C, Y is A; wherein A represents anoligopeptide, the sequence of which comprises a sequence which rendersthe ecPNA able to enter the nucleus of a cell; wherein B represents apeptide nucleic acid (PNA) structure at least 12 nucleotides in length,the sequence of which is capable of hybridizing with a DNA within thenucleus of the cell, which DNA is within a promoter region of a gene;wherein C represents an oligopeptide; and wherein each — represents achemical linkage between the structures at each side thereof, which maybe the same as or different from each other such linkage.
 2. The ecPNAof claim 1, wherein X is A and Y is C.
 3. The ecPNA of claim 1, whereinX is C and Y is A.
 4. (canceled)
 5. The ecPNA of claim 1, wherein theoligopeptide C is detectable when the ecPNA is bound to the DNA.
 6. TheecPNA of claim 1, wherein the sequence of C comprises a sequence whichrenders the ecPNA able to regulate the transcription and expression ofthe gene.
 7. The ecPNA of claim 1, wherein each — may be a chemical bondor a chemical linker.
 8. The ecPNA of claim 7, wherein at least one — isa chemical bond.
 9. The ecPNA of claim 8, wherein the chemical bond is acovalent bond, an amide bond, or a peptide bond.
 10. The ecPNA of claim7, wherein at least one — is a chemical linker.
 11. The ecPNA of claim10, wherein the chemical linker comprises an amino acid, biotin, anether (O), a stable polyether (OO), AEEA (2-aminoethoxy-2-ethoxyaceticacid), or a cleavable disulfide linkage. 12-15. (canceled)
 16. The ecPNAof claim 1, wherein A comprises a sequence selected from the groupconsisting of the following sequences: YGRKKRRQRRR (SEQ ID NO: 6),GRKERRQRRRPPQ (SEQ ID NO: 7), YARKARRQARR (SEQ ID NO: 8), YARAAARARA(SEQ ID NO: 9), YARAARRAARR (SEQ ID NO: 10), YARAARRAARA (SEQ ID NO:11), PKKKRKV (SEQ ID NO: 12), RQIKIWFQNRRMKWKK (SEQ ID NO: 13),KKWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKNKK (SEQ ID NO: 15),RQIKIWFPNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ ID NO: 17),RQIKIWFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRNRWRR (SEQ ID NO 19),RRWRRWWRRWWRRWR (SEQ ID NO: 20), RQILIWFQNRRMKWKK (SEQ ID NO: 22),LLIILRRRIRKQAHAHSK (SEQ ID NO: 23), KLALKLALKALKAALKLA (SEQ ID NO: 24),and AGYLLGKINLKALAALAKKIL (SEQ ID NO: 25).
 17. The ecPNA of claim 1,wherein C comprises a sequence selected from the group consisting of thefollowing sequences: DFDLDMLGDFDLDMLG (SEQ ID NO: 26), MLGDFDLDMLGDFDLD(SEQ ID NO: 30), CGSDALDDFDLDML (SEQ ID NO: 27), PEFPGIELLQELQALLQQ (SEQID NO: 28), and RHGEKWFLDDFTNNQMDQDY (SEQ ID NO: 29).
 18. The ecPNA ofclaim 1, wherein C comprises a sequence selected from the groupconsisting of the sequence of an engrailed repression domain, thesequence of the HID (HDAC interaction domain) of the Sin3A protein, andMSRRKQSKPRQIL (SEQ ID NO: 21).
 19. (canceled)
 20. The ecPNA of claim 1,wherein the gene is a γ-globin gene.
 21. The ecPNA of claim 3, whereinthe PNA structure comprises the sequence TACTCTAAGACTATT (PNA78) (SEQ IDNO: 1). 22-26. (canceled)
 27. A method of detecting the presence of aDNA within the nucleus of a cell which comprises contacting the cellwith the ecPNA of claim 1 under conditions such that the ecPNA entersthe cell and the PNA structure B hybridizes to the DNA to form ahybridization product, and then detecting the resulting hybridizationproduct.
 28. A method for upregulating transcription of a γ-globin genein a mammalian bone marrow cell comprising contacting the cell with theecPNA of claim
 20. 29. (canceled)
 30. A method for treating a β-globindisorder in a mammal comprising administering to said mammal atherapeutically effective amount of the ecPNA of claim
 20. 31-35.(canceled)
 36. A method for generating an inducible pluripotent stem(iPS) cell from a source cell comprising administering to the sourcecell an amount effective for inducing the generation of the iPS cell ofan ecPNA of claim 1, wherein the ecPNA upregulates transcription of agene selected from the group consisting of the following genes: OCT4,SOX2, c-NYC, KLF4, LIN28, NANOG, PRDM14, and NFRKB. 37-45. (canceled)46. An ecPNA having the general structure:H₂N—X—B—Y—COOH wherein X is D or C and Y is D or C with the proviso thatwhen X is D, Y is C, and when X is C, Y is D; wherein D represents acompound which renders the ecPNA able to bind to a receptor on a cell;wherein B represents a peptide nucleic acid (PNA) structure at least 12nucleotides in length, the sequence of which is capable of hybridizingwith a DNA within the nucleus of the cell, which DNA is within apromoter region of a gene; wherein C represents an structure; andwherein each — represents a chemical linkage between the structures ateach side thereof, which may be the same as or different from each othersuch linkage.
 47. The ecPNA of claim 1, wherein A comprises a sequenceselected from the group consisting of the following sequences:YGRKKRRQRRR (SEQ ID NO: 6), GRKKRRORRRPPQ (SEQ ID NO: 7), YARKARRQARR(SEQ ID NO: 8), YARAAARQARA (SEQ ID NO: 9), YARAARRAARR (SEQ ID NO: 10),YARAARRAARA (SEQ ID NO: 11), PKKKRKV (SEQ ID NO: 12), RQIKIWFQNRRMKWKK(SEQ ID NO: 13), KKWKMRRNQFWIKIQR (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQID NO: 15), RQIKIWFQNRRMKWKK (SEQ ID NO: 16), RQPKIWFPNRRMPWKK (SEQ IDNO: 17), RQIKINFQNMRRKWKK (SEQ ID NO: 18), RQIRIWFQNRRNRWRR (SEQ ID NO19), RRWRRWWRRWWRRWR (SEQ ID NO: 20), RQILIWFQNRRMKWKK (SEQ ID NO: 22),LLIILRRRIRKQAHAHSK (SEQ ID NO: 23), KLALKLALKALKAALKLA (SEQ ID NO: 24),and AGYLLGKINLKALAALAKKIL (SEQ ID NO: 25); wherein C represents anoligopeptide the sequence of which comprising a sequence selected fromthe group consisting of the following sequences: DFDLDMLGDFDLDMLG (SEQID NO: 26), MLGDFDLDMLGDFDLD (SEQ ID NO: 30), CGSDALDDFDLDML (SEQ ID NO:27), PEFPGIELQELELQELQALLQQ (SEQ ID NO: 28), RHGEKNFLDDFTNNQMDQDY (SEQID NO: 29), an engrailed repression domain, the sequence of the HID(HDAC interaction domain) of the Sin3A protein, and MSRRKQSKPRQIL (SEQID NO: 21); and wherein the PNA structure comprises the sequenceTACTCTAAGACTATT (SEQ ID NO: 1).